Stabilized polymeric thiol reagents

ABSTRACT

Disclosed are water soluble polymeric reagents comprising the structure POLY-[Y—S—W] x , where POLY is a water soluble polymer; Y is a hydrocarbon-based spacer group, x is 1 to 25, and S—W is a thiol, protected thiol, or thiol-reactive derivative. Preferably, the water soluble polymer is a PEG polymer. Also disclosed are conjugates of such polymeric reagents with pharmaceutically relevant molecules, and methods of their formation and use.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/316,051, filed Dec. 21, 2005, now U.S. Pat. No. 7,851,565, whichclaims priority to U.S. Provisional Application Ser. No. 60/639,823,filed Dec. 21, 2004, and U.S. Provisional Application Ser. No.60/705,968, filed Aug. 4, 2005, each of which is hereby incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to stabilized polymeric thiol reagentsderived from water-soluble polymers such as polyethylene glycol. Inparticular, the invention relates to the polymeric thiol reagents,conjugates thereof, and methods for utilizing such conjugates.

BACKGROUND OF THE INVENTION

Due to recent advances in biotechnology, therapeutic proteins and otherbiomolecules, e.g. antibodies and antibody fragments, can now beprepared on a large scale, making such biomolecules more widelyavailable. Unfortunately, the clinical usefulness of potentialtherapeutic biomolecules in unmodified form is often hampered by theirrapid proteolytic degradation, instability upon manufacture, storage oradministration, or by their immunogenicity. These deficiencies can oftenbe overcome by covalent attachment of a water-soluble polymer, such aspolyethylene glycol (PEG). See, for example, Abuchowski, A. et al., J.Biol. Chem. 252(11):3579 (1977); Davis, S. et al., Clin. Exp Immunol.46:649-652 (1981). The biological properties of PEG-modified proteins,also referred to as PEG-conjugates or PEGylated proteins, have beenshown, in many cases, to be considerably improved over those of theirnon-PEGylated counterparts (Herman et al., Macromol. Chem. Phys.195:203-209 (1994)). Polyethylene glycol-modified proteins have beenshown to possess longer circulatory times in the body, due to increasedresistance to proteolytic degradation, and also to possess increasedthermostability (Abuchowski, A. et al., J. Biol. Chem. 252:3582-3586(1977). A similar increase in bioefficacy is observed with otherbiomolecules, e.g. antibodies and antibody fragments (Chapman, A., Adv.Drug Del. Rev. 54:531-545 (2002)).

Polyethylene glycol having activated end groups suitable for reactionwith amino groups are commonly used for modification of proteins. Suchactivated PEGs or “polymeric reagents” include PEG-aldehydes (Harris, J.M. and Herati, R. S., Polym Prepr. (Am. Chem. Soc., Div. Polym. Chem.)32(1):154-155 (1991)), mixed anhydrides, N-hydroxysuccinimide esters,carbonylimidazolides, and chlorocyanurates (Herman, S. et al., Macromol.Chem. Phys. 195:203-209 (1994)). In some cases, however, polymerattachment through protein amino groups can be undesirable, such as whenderivatization of specific lysine residues inactivates the protein(Suzuki, T. et al., Biochimica et Biophysica Acta 788:248-255 (1984)).Therefore, it would be advantageous to have additional methods for themodification of a protein by PEG using another target amino acid forattachment, such as cysteine. Attachment to protein thiol groups oncysteine offers an advantage in that cysteines are typically lessabundant in proteins than lysines, thus reducing the likelihood ofprotein deactivation upon conjugation to these thiol-containing aminoacids. Thiol-selective activated polymers are described, for example, incommonly owned PCT publication no. WO 2004/063250.

Polymeric thiol derivatives, and specifically PEG thiols, are one typeof thiol-selective activated polymer. However, many prior art polymericthiols suffer from being highly susceptible to oxidative coupling toform disulfides, a degradative process that reduces the active componentand adds difficult-to-remove impurities. The latter can be particularlyproblematic in the preparation of bioconjugates from these materials.Therefore, it would be advantageous to provide polymeric thiol reagentshaving enhanced stability over prior art reagents.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a water-soluble activated polymer,also referred to as a “polymeric reagent,” having the structurePOLY-[Y—S—W]_(x)wherein:

-   -   POLY is a water soluble polymer;    -   Y is a divalent linking group, containing at least four carbon        atoms, consisting of a saturated or unsaturated hydrocarbon        backbone which is three to eight carbon atoms in length and has        substituents which are independently selected from hydrogen,        lower alkyl, lower alkenyl, and non-interfering substituents as        defined herein, where two such alkyl and/or alkenyl substituents        on different carbon atoms of the backbone may be linked so as to        form a cycloalkyl, cycloalkenyl, or aryl group;    -   S is a sulfur atom attached to an sp³ hybridized carbon of Y;    -   x is 1 to 25; and    -   S—W is a thiol, protected thiol, or thiol-reactive thiol        derivative. In one embodiment, S—W is a thiol-reactive        derivative, such as ortho-pyridyl disulfide (OPSS).

When x is 2, the reagent is a difunctional polymeric reagent, such asdescribed further below, and it may have a linear or a “forked”morphology, as described herein. The polymeric reagent may also have a“multiarmed” morphology, as described herein, particularly when x is 3or greater. In selected embodiments, x is 1 to 8, 1 to 6, or 1 to 4; infurther embodiments, x is 1 or 2, or x is 1. The POLY component of thedisclosed polymeric reagents can itself have a morphology selected fromthe group consisting of linear, branched, multi-armed, and combinationsthereof, as described further herein.

In a particular embodiment, when POLY is a linear polyethylene glycoland Y is a linear alkyl chain, POLY has a molecular weight of at least500.

In further embodiments, POLY has a molecular weight of at least 1000, orat least 2000. As an upper range, POLY has a molecular weight of notgreater than 300,000 Da.

The “hydrocarbon backbone” of the linking group Y is more particularlydefined as the shortest contiguous carbon chain connecting POLY and S.When the backbone of Y is unsaturated, it is preferably monounsaturated,i.e. having a single double or triple carbon-carbon bond. Preferably,the spacer group Y, including backbone and substituents, ismonounsaturated or, more preferably, fully saturated. In anotherembodiment, the spacer group Y, including backbone and substituents,consists of saturated and aromatic portions.

Preferably, the backbone is saturated. For example, Y may be of the form—(CR¹R²)_(n)—, where n is 3 to 8, and each of R¹ and R² is independentlyselected from hydrogen, lower alkyl, lower alkenyl, and anon-interfering substituent, where two groups R¹ and R² on differentcarbon atoms may be linked to form a cycloalkyl or aryl group. Inselected embodiments, n is 3 to 6, n is 4 to 6, or n=4, and each of R¹and R² is independently selected from hydrogen and lower alkyl, wherelower alkyl is preferably methyl or ethyl.

In further selected embodiments, Y is selected from the group consistingof C₄-C₈ alkylene, C₅-C₈ cycloalkylene, and combinations thereof, any ofwhich may include one or more non-interfering substituents.

Preferably, at most one or two non-interfering substituents, selectedfrom the group consisting of C₃-C₆ cycloalkyl, halo, cyano, loweralkoxy, and phenyl, and preferably selected from methoxy, ethoxy,fluoro, and chloro, are included. In one embodiment, noheteroatom-containing substituents are present; that is, the linking Yconsists of carbon and hydrogen.

In one preferred embodiment, each of R¹ and R² is hydrogen with respectto the n iterations of —(CR¹R²)—; in another preferred embodiment, eachof R¹ and R² is hydrogen with the exception of R¹ on a carbon adjacentsaid sulfur atom, said R¹ being lower alkyl, preferably methyl or ethyl(α-branching). In one embodiment, the α-branch group is methyl.

In embodiments of Y where Y is —(CR¹R²)_(n)— and two groups R¹ and R² ondifferent carbon atoms are linked to form a cycloalkyl, cycloalkenyl, oraryl group, the cycloalkyl group is preferably a cyclopentyl orcyclohexyl group. Preferably, S is linked to an sp³ hybridized acycliccarbon of Y in such embodiments.

As noted above, POLY may be a polyethylene glycol (PEG). Such a PEGtypically has a molecular weight of 148 (e.g. a trimer plus linkingoxygen atom) to about 200 to about 100,000 Daltons. In selectedembodiments, the polyethylene glycol has a molecular weight from about200 to about 40,000 Daltons. Representative molecular weights include,for example, 500, 1000, 2000, 2500, 3500, 5000, 7500, 10000, 15000,20000, 25000, 30000, and 40000 Daltons. The PEG component of thedisclosed reagents can itself have a morphology selected from the groupconsisting of linear, branched, multi-armed, and combinations thereof,as described further herein.

Accordingly, the invention provides a water soluble polymeric reagentcomprising the structure:PEG-[Y—S—W]_(x)wherein:

-   -   PEG is a polyethylene glycol polymer;    -   Y is a divalent linking group consisting of a saturated or        unsaturated hydrocarbon backbone which is three to eight carbon        atoms in length and has substituents which are independently        selected from hydrogen, lower alkyl, lower alkenyl, and        non-interfering substituents as defined herein, where two such        alkyl and/or alkenyl substituents on different carbon atoms of        the backbone may be linked so as to form a cycloalkyl,        cycloalkenyl, or aryl group;    -   S is a sulfur atom attached to an sp³ hybridized carbon of Y;    -   x is 1 to 25;    -   S—W is a thiol, protected thiol, or thiol-reactive thiol        derivative; and    -   PEG has a molecular weight of at least 500 when PEG is linear, x        is 1, and Y is a linear alkyl chain.

As stated above, when x is 2, the reagent is a difunctional polymericreagent, such as described further below, and it may have a linear or a“forked” morphology, as described herein. The polymeric reagent may alsohave a “multiarmed” morphology, as described herein, particularly when xis 3 or greater. In selected embodiments, x is 1 to 8, 1 to 6, or 1 to4; in further embodiments, x is 1 or 2, or x is 1. The PEG component ofthe disclosed reagents can itself have a morphology selected from thegroup consisting of linear, branched, multi-armed, and combinationsthereof, as described further herein.

In preferred embodiments, PEG has a molecular weight of at least 148, atleast 200, at least 500, at least 1000, or at least 2000, up to about100,000 Daltons, including the various ranges noted above, and amorphology selected from linear, branched, forked, and multiarmed. S—Wis preferably a thiol-reactive thiol derivative, and more preferablyortho-pyridyl disulfide (OPSS).

The “hydrocarbon backbone” of the linking group Y is more particularlydefined as the shortest contiguous carbon chain connecting PEG and S.Preferably, the backbone of the linking group Y is saturated, such thatY has the formula —(CR¹R²)_(n)—, where n is 3 to 8, and each of R¹ andR² is independently selected from hydrogen, lower alkyl, lower alkenyl,and a non-interfering substituent, where two groups R¹ and R² ondifferent carbon atoms may be linked to form a cycloalkyl or aryl group.In selected embodiments, n is 3 to 6, n is 4 to 6, or n=4, and each ofR¹ and R² is independently selected from hydrogen and methyl.

In further selected embodiments, Y is selected from the group consistingof C₃-C₈ alkylene, C₅-C₈ cycloalkylene, and combinations thereof, any ofwhich may include one or more non-interfering substituents, as describedabove. Preferably, at most one or two non-interfering substituents,selected from the group consisting of C₃-C₆ cycloalkyl, halo, cyano,lower alkoxy, and phenyl, and preferably selected from methoxy, ethoxy,fluoro, and chloro, are included. In one embodiment, noheteroatom-containing substituents are present; that is, the linkinggroup Y consists of carbon and hydrogen.

As above, in one preferred embodiment, each of R¹ and R² is hydrogenwith respect to the n iterations of —(CR¹R²)—; in another preferredembodiment, each of R¹ and R² is hydrogen with the exception of R¹ on acarbon adjacent said sulfur atom, said R¹ being lower alkyl, preferablymethyl or ethyl (α-branching). In one embodiment, the α-branch group ismethyl.

In embodiments of Y where Y is —(CR¹R²)_(n)— and two groups R¹ and R² ondifferent carbon atoms are linked to form a cycloalkyl, cycloalkenyl, oraryl group, the cycloalkyl group is preferably a cyclopentyl orcyclohexyl group. Preferably, S is linked to an sp³ hybridized acycliccarbon of Y in such embodiments.

In an exemplary reagent of the form PEG-Y—S—W, Y is —(CR¹R²)_(n)— whereeach of R¹ and R² is hydrogen and n is 4, S—W is ortho-pyridyl disulfide(OPSS), and PEG is a methoxy-terminated polyethylene glycol (mPEG). ThemPEG preferably has a molecular weight in the range of 5000 to 30000 Da.In further exemplary reagents, PEG and SW are similarly defined, n is 3or 4, and each of R¹ and R² is hydrogen with the exception of R¹ on acarbon adjacent said sulfur atom, said R¹ being methyl (i.e., Y is—CH₂CH₂CH(CH₃)— or —CH₂CH₂CH₂CH(CH₃)—).).

The water soluble polymeric reagents may have a polyfunctionalstructure, as shown:POLY-[Y—S—W]_(x)wherein:

-   -   POLY is a water soluble polymer;    -   x is 2 to 25;    -   each Y is a divalent linking group, having at least four carbon        atoms, consisting of a saturated or unsaturated hydrocarbon        backbone which is three to ten, preferably three to eight,        carbon atoms in length and has substituents which are        independently selected from hydrogen, lower alkyl, lower        alkenyl, and non-interfering substituents as defined herein,        where two such alkyl and/or alkenyl substituents on different        carbon atoms of the backbone may be linked so as to form a        cycloalkyl, cycloalkenyl, or aryl group;    -   each S is a sulfur atom attached to an sp³ hybridized carbon of        the adjacent Y; and    -   each S—W is a independently a thiol, protected thiol, or        thiol-reactive thiol derivative.

Preferably, the two or more Y groups are identical; the two or more Wgroups are also typically identical. Alternatively, particularly in adifunctional reagent (x=2), the two SW's may be different; e.g. one SWis a thiol or protected thiol while the other is a thiol-reactivederivative, or one SW is a thiol or thiol-reactive derivative while theother is a protected thiol.

As noted above, when x is 2, the polymeric reagent may have a linear ora “forked” morphology, as described herein. The polymeric reagent mayalso have a “multiarmed” morphology, as described herein, particularlywhen x is 3 or greater. In selected embodiments, x is 2 to 8, 2 to 6, or2 to 4; in one embodiment, x is 2. The POLY component of the disclosedreagents can itself have a morphology selected from the group consistingof linear, branched, multi-armed, and combinations thereof, as describedfurther herein.

As above, the backbone of Y is preferably saturated, such that each Y isa linker having at least four carbon atoms and having the formula—(CR¹R²)_(n)—, where n is 3 to 10, preferably 3 to 8, and each of R¹ andR² is independently selected from hydrogen, lower alkyl, lower alkenyl,and a non-interfering substituent, where two groups R¹ and R² ondifferent carbon atoms may be linked to form a cycloalkyl or aryl group.Further preferred embodiments of Y, and of POLY, are generally asdefined above for the monomeric reagent POLY-Y—S—W.

The corresponding PEG-based polyfunctional polymeric reagents have thestructure:PEG-[Y—S—W]_(x)wherein:

-   -   PEG is polyethylene glycol polymer;    -   x is 2 to 25;    -   each Y is a divalent linking group consisting of a saturated or        unsaturated hydrocarbon backbone which is three to ten,        preferably three to eight, carbon atoms in length and has        substituents which are independently selected from hydrogen,        lower alkyl, lower alkenyl, and non-interfering substituents as        defined herein, where two such alkyl and/or alkenyl substituents        on different carbon atoms of the backbone may be linked so as to        form a cycloalkyl, cycloalkenyl, or aryl group;    -   each S is a sulfur atom attached to an sp³ hybridized carbon of        the adjacent Y; and    -   each S—W is a independently a thiol, protected thiol, or        thiol-reactive thiol derivative.

Preferably, the multiple Y groups are identical; the multiple W groupsare also typically identical. The backbone of Y is preferably saturated,such that each Y is a linker having the formula —(CR¹R²)_(n)—, where nis 3 to 10, preferably 3 to 8, and each of R¹ and R² is independentlyselected from hydrogen, lower alkyl, lower alkenyl, and anon-interfering substituent, where two groups R¹ and R² on differentcarbon atoms may be linked to form a cycloalkyl or aryl group.

Further preferred embodiments of Y, and of PEG, are generally as definedabove for PEG-[Y—S—W]_(x). In an exemplary difunctional reagent of theform W—S—Y-PEG-Y—S—W, each Y is —(CR¹R²)n— where each of R¹ and R² ishydrogen and n is 4, S—W is ortho-pyridyl disulfide (OPSS), and each PEGis a methoxy-terminated polyethylene glycol (mPEG). The mPEG preferablyhas a molecular weight in the range of 1000 to 5000 Da, e.g. about 2000or about 3400 Da. In a further exemplary reagent, PEG and SW aresimilarly defined, n is 3 or 4, and each of R¹ and R² is hydrogen withthe exception of R¹ on a carbon adjacent said sulfur atom, said R¹ beingmethyl.

In a related aspect, the invention provides a polymer conjugatecomprising the structure:POLY-[Y—S—S-A]_(x)wherein:

-   -   POLY is a water soluble polymer;    -   x is 1 to 25;    -   Y is a divalent linking group consisting of a saturated or        unsaturated hydrocarbon backbone which is three to ten,        preferably three to eight, carbon atoms in length and has        substituents which are independently selected from hydrogen,        lower alkyl, lower alkenyl, and non-interfering substituents as        defined herein, where two such alkyl and/or alkenyl substituents        on different carbon atoms of the backbone may be linked so as to        form a cycloalkyl, cycloalkenyl, or aryl group;    -   S—S is a disulfide group attached to an sp³ hybridized carbon of        Y; and    -   A is a covalently linked residue (as defined herein) of a        pharmacologically active molecule.

In selected embodiments, x is 1 to 8, 1 to 6, or 1 to 4; in furtherembodiments, x is 1 or 2, or x is 1. When x is 2, the conjugate may havea linear or a “forked” morphology, as described herein. The conjugatemay also have a “multiarmed” morphology, as described herein,particularly when x is 3 or greater. The POLY component of the conjugatecan itself have a morphology selected from the group consisting oflinear, branched, multi-armed, and combinations thereof, as describedfurther herein.

Preferably, the multiple Y groups are identical. The hydrocarbonbackbone of Y is preferably saturated, with Y having the formula—(CR¹R²)n—, where n is 3 to 10, preferably 3 to 8, each of R¹ and R² isindependently selected from hydrogen, alkyl, alkenyl, and anon-interfering substituent, and where two groups R¹ and R² on differentcarbon atoms may be linked to form a cycloalkyl or aryl group. Morepreferably, Y is selected from the group consisting of C₃-C₈ alkylene,C₅-C₈ cycloalkylene, aryl, and combinations thereof, any of which mayinclude one or more non-interfering substituents. In one embodiment, Yhas at least four carbon atoms.

In further embodiments, Y is a linear or branched alkylene having theformula —(CR¹R²)n—, where n is 3 to 10, and each of R¹ and R² isindependently selected from hydrogen, lower alkyl, lower alkenyl, and anon-interfering substituent. More preferably, n is 3 to 8, or 3 to 6,and each of R¹ and R² is independently selected from hydrogen andmethyl. In selected embodiments, each of R¹ and R² is hydrogen. Inanother preferred embodiment, each of R¹ and R² is hydrogen with theexception of R¹ on a carbon adjacent said disulfide linkage, said R¹being lower alkyl, e.g. methyl or ethyl.

Other embodiments include those in which Y is —(CR¹R²)n—, where n is 3to 10, preferably 3 to 8, and two groups R¹ and R² on different carbonatoms are linked to form a cycloalkyl or aryl group, preferably acycloalkyl such as cyclopentyl or cyclohexyl group.

The water soluble polymer POLY preferably has a molecular weight of atleast 500, or at least 1000. The molecular weight of POLY is typicallygreater than 200 and less than about 300K Daltons, preferably less thanabout 200K Daltons, and more preferably less than about 100K Daltons. Inone embodiment, POLY is a polyethylene glycol, preferably having amolecular weight of 148 to about 200 to about 100,000 Daltons, and amorphology selected from linear, branched, forked, and multiarmed. Inselected embodiments, the polyethylene glycol has a molecular weightfrom about 200 to about 40,000 Daltons. Representative molecular weightsinclude, for example, 500, 1000, 2500, 3500, 5000, 7500, 10000, 15000,20000, 25000, 30000, and 40000 Daltons.

The molecule conjugated to the water soluble polymer, represented by Ain its conjugated form, has a reactive thiol group in its unconjugatedform and is preferably selected from the group consisting of proteins,peptides, and small molecules, typically small organic molecules.

The conjugate is preferably itself water soluble. The conjugate may beprovided in or with a suitable pharmaceutical excipient, such as anaqueous carrier, for therapeutic use.

In a related aspect, the invention provides a method for delivering apharmacologically active molecule having a reactive thiol group to asubject, by administering to the subject a conjugate as described above,in a pharmaceutically acceptable carrier. The conjugate is typicallyprepared by conjugating the molecule with any of the water solublepolymeric reagents described herein.

The hydrocarbon-based segment(s), Y, in the activated polymeric reagentsof the invention, being hydrophobic in nature, are effective to reducethe tendency towards dimerization of these reagents, relative to priorart reagents in which the thiol is linked to a heteroatom in the polymersegment (or in a linking moiety) by, for example, a two-carbon linkage.Branching of Y at the carbon adjacent to the sulfur atom (α-branching)is further effective to reduce dimerization. The hydrocarbon-basedsegment Y also reduces cleavage, e.g. enzymatic cleavage in vivo, of theadjacent disulfide bond, in conjugates formed using these reagents.

The reagents disclosed herein are further characterized as being“linkerless” reagents; that is, the water-soluble polymer, preferably aPEG, is directly linked to the hydrocarbon-based spacer group Y. Theabsence of heteroatom-containing linkages, such as amides, esters, orcarbamates, between the active conjugating functionality, i.e. the thiolor protected thiol, and the polymer reduces the potential fordegradation of the resulting conjugate. Moreover, the presence of theselinkages, such as amides, in such reagents can trigger a deleteriousimmune response. This potential is eliminated or greatly reduced by useof the current “linkerless” reagents.

As shown in Example 2 herein, a polymeric thiol reagent of the inventionwas more stable under synthetic conditions than a corresponding reagenthaving only a two-carbon linker between the hydrophilic polymer (PEG)and thiol group. This increased stability is also exhibited in Example 9and comparative Example 10. The conjugation behavior of the polymericthiol reagent of the invention was similar to that observed for apolymeric reagent (a maleimide-terminated polymer) known not todimerize, as also shown in Example 2.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a PAGE analysis of a conjugation reaction between apolymeric reagent of the invention, designated mPEG₅₀₀₀-4C-OPSS, asdescribed in Examples 1-2, with reduced BSA, and the correspondingconjugation reaction of the polymeric reagent mPEG₅₀₀₀-maleimide withreduced BSA: Lane 1, standards; Lane 2, reduced BSA; Lane 3, conjugationwith mPEG-MAL; Lane 4, conjugation with mPEG-4C-OPSS. The gel is stainedwith SimplyBlue Safe Stain; and

FIG. 2 shows the gel of FIG. 1 additionally stained with Barium Iodine,for detection of PEG.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The following terms as used herein have the meanings indicated. As usedin the specification, and in the appended claims, the singular forms“a,” “an,” “the,” include plural referents unless the context clearlydictates otherwise.

“PEG” or “polyethylene glycol,” as used herein, is meant to encompassany water soluble polyethylene oxide). Typically, PEGs for use in thepresent invention will comprise one of the two following structures:—CH₂CH₂—O—(CH₂CH₂O)_(m)—CH₂CH₂— or —O(CH₂CH₂O)_(m)—, where m isgenerally from 2 to about 6000, more typically 4 or 5 to about 2500. Ina broader sense, “PEG” can refer to a polymer that contains a majority,i.e., greater than 50%, of subunits that are —CH₂CH₂O—. Preferably,greater than 75%, greater than 95%, or substantially all of themonomeric subunits are —CH₂CH₂O—. The terminal groups and architectureof the overall PEG may vary. One terminus of the PEG may contain anend-capping group, which is generally a carbon-containing groupcomprised of 1-20 carbons and is preferably selected from alkyl,alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heterocyclo, andsubstituted forms of any of the foregoing. The end-capping group canalso be a silane. An end-capping group is one that does not readilyundergo chemical transformation under typical synthetic reactionconditions. Most preferred are alkyl (alkoxy) or aralkyl (aralkoxy)capping groups, such as methyl, ethyl or benzyl.

The end-capping group can also advantageously comprise a phospholipid.When the polymer has an end-capping group such as a phospholipid, uniqueproperties (such as the ability to form organized structures withsimilarly end-capped polymers) are imparted to the polymer. Exemplaryphospholipids include, without limitation, those selected from the classof phospholipids called phosphatidylcholines. Specific phospholipidsinclude, without limitation, those selected from the group consisting ofdilauroyl phosphatidylcholine, dioleyl phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoyl phosphatidylcholine, behenoylphosphatidylcholine, arachidoyl phosphatidylcholine, and lecithin.

The end-capping group can also advantageously comprise a detectablelabel. Such labels include, without limitation, fluorescers,chemiluminescers, moieties used in enzyme labeling, colorimetric (e.g.,dyes), metal ions, radioactive moieties, and the like.

The PEG may also be terminated with a functional group, such as thosedescribed below, preferably in protected form.

Specific PEG forms for use in the invention include PEGs having avariety of molecular weights, structures or geometries (e.g., branched,linear, forked, multiarmed).

“Branched,” in reference to the geometry or overall structure of apolymer, refers to polymer having 2 or more polymer “arms.” A branchedpolymer may possess 2 polymer arms, 3 polymer arms, 4 polymer arms, 6polymer arms, 8 polymer arms or more. One particular type of highlybranched polymer is a dendritic polymer or dendrimer, that for thepurposes of the invention, is considered to possess a structure distinctfrom that of a branched polymer. A “branch point” refers to abifurcation point comprising one or more atoms at which a polymer splitsor branches from a linear structure into one or more additional polymerarms.

A “dendrimer” is a globular, size monodisperse polymer in which allbonds emerge radially from a central focal point or core with a regularbranching pattern and with repeat units that each contribute a branchpoint. Dendrimers exhibit certain dendritic state properties such ascore encapsulation, making them unique from other types of polymers.

“Water-soluble,” in the context of a polymer of the invention or a“water-soluble polymer segment”, is any segment or polymer that issoluble in water at room temperature. Typically, a water-soluble polymeror segment will transmit at least about 75%, more preferably at leastabout 95% of light, transmitted by the same solution after filtering. Ona weight basis, a water-soluble polymer or segment thereof willpreferably be at least about 35% (by weight) soluble in water, morepreferably at least about 50% (by weight) soluble in water, still morepreferably about 70% (by weight) soluble in water, and still morepreferably about 85% (by weight) soluble in water. It is most preferred,however, that the water-soluble polymer or segment is about 95% (byweight) soluble in water or completely soluble in water.

“Molecular mass” or “molecular weight” of a polymer, unless otherwisespecified, refers to number average molecular weight. Number averagemolecular weight (M_(n)) is defined as ΣN_(i)M_(i)/ΣN_(i), wherein N_(i)is the number of polymer molecules (or the number of moles of thosemolecules) having molecular weight M_(i). The number average molecularweight of a polymer can be determined by osmometry, end-group titration,and colligative properties. Weight average molecular weight is definedas ΣN_(i)M_(i) ²/ΣN_(i)M_(i), where N_(i) is again the number ofmolecules of molecular weight M_(i). The weight average molecular weightcan be determined by light scattering, small angle neutron scattering(SANS), X-ray scattering, and sedimentation velocity.

The polymers of the invention, or employed in the invention, may bepolydisperse; i.e., the number average molecular weight and weightaverage molecular weight of the polymers are not equal. However, thepolydispersity values, expressed as a ratio of weight average molecularweight (M_(w)) to number average molecular weight (M_(n)),(M_(w)/M_(n)), are generally low; that is, less than about 1.2,preferably less than about 1.15, more preferably less than about 1.10,still more preferably less than about 1.05, yet still most preferablyless than about 1.03, and most preferably less than about 1.025.

The term “reactive” or “activated” refers to a functional group thatreacts readily or at a practical rate under conventional conditions oforganic synthesis. This is in contrast to those groups that either donot react or require strong catalysts or impractical reaction conditionsin order to react (i.e., a “nonreactive” or “inert” group).

“Not readily reactive” or “inert,” with reference to a functional grouppresent on a molecule in a reaction mixture, indicates that the groupremains largely intact under conditions effective to produce a desiredreaction in the reaction mixture.

A “protecting group” is a moiety that prevents or blocks reaction of aparticular chemically reactive functional group in a molecule undercertain reaction conditions. The term may also refer to the protectedform of a functional group. The protecting group will vary dependingupon the type of chemically reactive group being protected as well asthe reaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule. Functional groups whichmay be protected include, by way of example, carboxylic acid groups,amino groups, hydroxyl groups, thiol groups, carbonyl groups and thelike. Representative protected forms of such functional groups include,for carboxylic acids, esters (such as a p-methoxybenzyl ester), amidesand hydrazides; for amino groups, carbamates (such astert-butoxycarbonyl or fluorenylmethoxycarbonyl) and amides; forhydroxyl groups, ethers and esters; for thiol groups, thioethers andthioesters; for carbonyl groups, acetals and ketals; and the like. Suchprotecting groups are well-known to those skilled in the art and aredescribed, for example, in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

A “thiol-reactive derivative” of a thiol refers to a thiol derivativewhich can react with another thiol, preferably under conditions ofmoderate temperature and neutral or physiological pH, to form adisulfide linkage. Preferably, the reaction forms only stablebyproducts. Typical examples of such derivatives are ortho-pyridyldisulfides and TNB-thiol derivatives (where TNB is 5-thio-2-nitrobenzoicacid). See e.g. Hermanson, Bioconjugate Techniques, Academic Press,1996, pp 150-152.

As used herein, the term “functional group” or any synonym thereof ismeant to encompass protected or derivatized forms thereof. Similarly,the term “thiol reagent” or “polymeric thiol” encompasses protected orderivatized thiol reagents or polymeric protected or derivatized thiols(such as polymer-OPSS).

A “physiologically cleavable” or “hydrolysable” or “degradable” bond isa relatively weak bond that reacts with water (i.e., is hydrolyzed)under physiological conditions. The tendency of a bond to hydrolyze inwater will depend not only on the general type of linkage connecting twocentral atoms but also on the substituents attached to these centralatoms. Appropriate hydrolytically unstable or weak linkages include butare not limited to carboxylate ester, phosphate ester, anhydrides,acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides andoligonucleotides, thioesters, thiolesters, and carbonates. An“enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95%, 99% or greater of some given quantity.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to20 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or, preferably, linear(unbranched). Exemplary alkyl groups include ethyl, propyl, butyl,pentyl, 2-methylbutyl, 2-methylpropyl (isobutyl), 3-methylpentyl, andthe like. As used herein, “alkyl” includes cycloalkyl when three or morecarbon atoms are referenced. “Alkylene” refers to a divalent alkylgroup, e.g. —(CH₂)_(x)—.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, more preferably 1 to 4 carbon atoms, as exemplified by methyl,ethyl, n-butyl, isopropyl, and t-butyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or spiro cyclic compounds, preferablymade up of 3 to about 12 carbon atoms, more preferably 3 to about 8.“Cycloalkylene” refers to a divalent cycloalkyl group.

As used herein, “alkenyl” refers to a branched or unbranched hydrocarbongroup having 2 to 15 carbon atoms and containing at least one doublebond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl,octenyl, decenyl, tetradecenyl, and the like.

The term “alkynyl” as used herein refers to a branched or unbranchedhydrocarbon group having 2 to 15 atoms and containing at least onetriple bond, such as ethynyl, n-propynyl, isopentynyl, n-butynyl,octynyl, decynyl, and so forth.

“Alkoxy” refers to an —OR group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy, etc.),more preferably lower alkyl (i.e. C₁-C₆ or C₁-C₄).

“Aryl” refers to a substituted or unsubstituted monovalent aromaticradical having a single ring (e.g., phenyl) or two condensed or fusedrings (e.g., naphthyl). Multiple aryl rings may also be unfused (e.g.biphenyl). The term includes heteroaryl groups, which are aromatic ringgroups having one or more nitrogen, oxygen, or sulfur atoms in the ring,such as furyl, pyrrole, pyridyl, and indole.

“Aralkyl” refers to an alkyl, preferably lower (C₁-C₄, more preferablyC₁-C₂) alkyl, substituent which is further substituted with an arylgroup; examples are benzyl and phenethyl. “Aralkoxy” refers to a groupof the form -OR where R is aralkyl; one example is benzyloxy.

A “heterocycle” refers to a ring, preferably a 5- to 7-membered ring,whose ring atoms are selected from the group consisting of carbon,nitrogen, oxygen and sulfur. Preferably, the ring atoms include 3 to 6carbon atoms. Examples of aromatic heterocycles (heteroaryl) are givenabove; non-aromatic heterocycles include, for example, pyrrolidine,piperidine, piperazine, and morpholine.

A “substituted” group or moiety is one in which a hydrogen atom has beenreplaced with a non-hydrogen atom or group, which is preferably anon-interfering substituent.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

These include, but are not limited to, lower alkyl, alkenyl, or alkynyl;lower alkoxy; C₃-C₆ cycloalkyl; halo, e.g., fluoro, chloro, bromo, oriodo; cyano; oxo (keto); nitro; and phenyl.

By “residue” is meant the portion of a molecule remaining after reactionwith one or more molecules. For example, a biologically active moleculeresidue in a polymer conjugate of the invention typically corresponds tothe portion of the biologically active molecule up to but excluding thecovalent linkage resulting from reaction of a reactive group on thebiologically active molecule with a reactive group on a polymer reagent.

The term “conjugate” refers to an entity formed as a result of covalentattachment of a molecule, e.g., a biologically active molecule, to areactive polymer molecule, preferably a poly(ethylene glycol).

Each of the terms “drug,” “biologically active molecule,” “biologicallyactive moiety,” “biologically active agent”, “pharmacologically activeagent”, and “pharmacologically active molecule”, when used herein, meansany substance which can affect any physical or biochemical property of abiological organism, where the organism may be selected from viruses,bacteria, fungi, plants, animals, and humans. In particular, as usedherein, biologically active molecules include any substance intended fordiagnosis, cure mitigation, treatment, or prevention of disease inhumans or other animals, or to otherwise enhance physical or mentalwell-being of humans or animals. Examples of biologically activemolecules include, but are not limited to, peptides, proteins, enzymes,small molecule drugs, dyes, lipids, nucleosides, oligonucleotides,polynucleotides, nucleic acids, cells, viruses, liposomes,microparticles and micelles. Classes of biologically active agents thatare suitable for use with the invention include, but are not limited to,antibiotics, fungicides, anti-viral agents, anti-inflammatory agents,anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones,growth factors, steroidal agents, and the like. Also included are foods,food supplements, nutrients, nutraceuticals, drugs, vaccines,antibodies, vitamins, and other beneficial agents.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to an excipient that can be included in the compositionsof the invention and that causes no significant adverse toxicologicaleffects to a patient.

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used herein to referto mean the amount of a polymer-active agent conjugate present in apharmaceutical preparation that is needed to provide a desired level ofactive agent and/or conjugate in the bloodstream or in the targettissue. The precise amount will depend upon numerous factors, e.g., theparticular active agent, the components and physical characteristics ofpharmaceutical preparation, intended patient population, patientconsiderations, and the like, and can readily be determined by oneskilled in the art, based upon the information provided herein andavailable in the relevant literature.

The term “patient” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of abiologically active agent or conjugate thereof, and includes both humansand animals.

Polymeric Thiol Reagents

The water-soluble, polymeric reagents of the invention comprise thestructurePOLY-[Y—S—W]_(x)wherein:

-   -   POLY is a water-soluble polymer segment;    -   x is 1 to 25;    -   Y is a divalent linking group comprising at least four carbon        atoms, and consisting of a saturated or unsaturated hydrocarbon        backbone which is three to eight carbon atoms in length and has        substituents which are independently selected from hydrogen,        lower alkyl, lower alkenyl, and non-interfering substituents as        defined herein, where two such alkyl and/or alkenyl substituents        on different carbon atoms of the backbone may be linked so as to        form a cycloalkyl, cycloalkenyl, or aryl group;    -   S is a sulfur atom attached to an sp³ hybridized carbon of Y;    -   and S—W is a thiol (i.e. W is H), protected thiol, or        thiol-reactive derivative.

In one embodiment, S—W is a thiol-reactive derivative, such asortho-pyridyl disulfide (OPSS). Protected thiols include, for example,thioethers, such as S-benzyl or S-trityl ethers, and thioesters.

The sulfur atom S is attached to an sp³ hybridized carbon atom of Y, asnoted, rather than to an aryl ring or double bond. In one embodiment,the carbon atom to which the sulfur atom is attached has a lower alkylsubstituent, such as methyl or ethyl (α-branching).

The “backbone” of the spacer group Y is more particularly defined as theshortest contiguous carbon chain connecting POLY and S. In oneembodiment, the backbone of Y is saturated. For example, Y may be of theform —(CR¹R²)_(n)—, where n is 3 to 8, preferably 3 to 6, each of R¹ andR² is independently selected from hydrogen, lower alkyl, lower alkenyl,and a non-interfering substituent, and where two groups R¹ and R² ondifferent carbon atoms of —(CR¹R²)_(n)— may be linked to form acycloalkyl, cycloalkenyl, or aryl group. When Y contains a cycloalkylgroup, it is preferably a five- or six-membered cycloalkyl group.

In selected embodiments, Y is selected from the group consisting ofC₃-C₅ alkylene and combinations of C₃-C₈ alkylene with C₅-C₈cycloalkylene or aryl, any of which may include one or morenon-interfering substituents. Preferably, at most one or twonon-interfering substituents, selected from the group consisting ofC₃-C₆ cycloalkyl, halo, cyano, lower alkoxy, and phenyl, and preferablyselected from methoxy, ethoxy, fluoro, and chloro, are included. In oneembodiment, no heteroatom-containing substituents are present; that is,Y consists of carbon and hydrogen.

When the backbone of Y is unsaturated, it is preferably monounsaturated,i.e. having a single double or triple carbon-carbon bond. Preferably,the spacer group Y, including backbone and substituents, ismonounsaturated or, more preferably, fully saturated. In thisembodiment, Y may be a fully saturated hydrocarbon.

In another embodiment, the spacer group Y, including backbone andsubstituents, consists of saturated and aromatic portions, preferablysaturated and aromatic hydrocarbon portions.

In the polymeric reagents, when Y is —(CR¹R²)_(n)—, the polymer segmentPOLY preferably has a molecular weight of at least 500 Da when each ofR¹ and R² is hydrogen with respect to the n iterations of —(CR¹R²)—,particularly when POLY is a linear PEG and x=1 in the formula above.POLY may further have a molecular weight greater than 500 Da, greaterthan 750 Da, or greater than 1000 Da. A variety of greater molecularweight ranges, up to about 300,000 Da, more typically up to about100,000 Da, can be used, as described above.

When x is 2, the reagent is a difunctional polymeric reagent, such asdescribed further below, and it may have a linear or a “forked”morphology, as described herein. The polymeric reagent may also have a“multiarmed” morphology, as described herein, particularly when x is 3or greater. In selected embodiments, x is 1 to 8, 1 to 6, or 1 to 4; infurther embodiments, x is 1 or 2, or x is 1. The POLY component of thedisclosed reagents can itself have a morphology selected from the groupconsisting of linear, branched, multi-armed, and combinations thereof,as described further herein.

In a preferred embodiment, the water soluble polymer segment is apolyethylene glycol, such that the reagent has the formulaPEG-[Y—S—W]_(x)wherein:

-   -   PEG is a poly(ethylene glycol);    -   Y is a divalent linking group consisting of a saturated or        unsaturated hydrocarbon backbone which is three to eight carbon        atoms in length and has substituents which are independently        selected from hydrogen, lower alkyl, lower alkenyl, and        non-interfering substituents as defined herein, where two such        alkyl and/or alkenyl substituents on different carbon atoms of        the backbone may be linked so as to form a cycloalkyl,        cycloalkenyl, or aryl group;    -   S is a sulfur atom attached to an sp³ hybridized carbon of Y;    -   x is 1 to 25; and    -   S—W is a thiol (i.e. W is H), protected thiol, or thiol-reactive        derivative.

The inventors have found that, by including a hydrophobic spacer groupbetween the water-soluble polymer segment and the thiol group in awater-soluble polymeric thiol, the tendency of such a molecule todimerize to form disulfides is reduced. Yields are accordingly increasedin preparation of such reagents and in their conjugation with othermolecules, as demonstrated below.

The spacer groups described herein are hydrocarbon-based groups morethree carbons or more in length, which preferably contain at least fourcarbon atoms, which may include branching carbons (e.g. an isobutylene,or 1-methylpropylene, linkage). Although described as “hydrocarbonbased”, the spacer group may include a limited number of non-interferingsubstituents as defined herein. Preferably, however, the spacer groupconsists of carbon and hydrogen.

In preferred embodiments of the polymeric reagent, Y is a linear orbranched alkylene having the formula —(CR¹R²)_(n)—, where n is 3 to 8,and each of R¹ and R² is independently selected from hydrogen, loweralkyl, lower alkenyl, and a non-interfering substituent. Preferably,zero to two, more preferably zero or one, such non-interferingsubstituents are included.

Preferably, n is 4 to 8, more preferably 4 to 6. In one embodiment, eachof R¹ and R² is independently selected from hydrogen and methyl. In apreferred embodiment, each of R¹ and R² is hydrogen with respect to then iterations of —(CR¹R²)—; in another preferred embodiment, each of R¹and R² is hydrogen with the exception of R¹ on a carbon adjacent saidsulfur atom (α-carbon), said R¹ being lower alkyl, preferably methyl orethyl (α-branching). In one embodiment, the α-branch group is methyl.

In embodiments of Y where Y is —(CR¹R²)_(n)—, n is 4 to 8, and twogroups R¹ and R² on different carbon atoms are linked to form acycloalkyl, cycloalkenyl, or aryl group, the cycloalkyl group ispreferably a cyclopentyl or cyclohexyl group. In such embodiments, thesulfur atom is preferably attached to a non-cyclic carbon of Y.

Exemplary spacer groups Y having a saturated backbone include thefollowing (where the curved lines indicate bonds to POLY or S, so thatthe first structure, for example, represents n-butylene):

As noted above, the “backbone” of the spacer group is the shortestcontiguous carbon chain linking POLY to the sulfur atom. Accordingly,each of the structures in the second row above has a five-carbonbackbone. By this definition, moreover, the last structure shown has asaturated backbone, although the spacer group as a whole is unsaturated.

In one exemplary polymeric reagent, depicted below, POLY ismethoxy-terminated polyethylene glycol (mPEG), Y is —(CH₂)₄—, and —S—Wis ortho-pyridyl disulfide (OPSS), as depicted below, or SH. The mPEGpreferably has a molecular weight in the range of 5000 to 30000 Da; e.g.about 5000, about 10000, about 20000, or about 30000 Da.

In a further embodiment, the polymeric reagent is a difunctionalstructure represented by W—S—Y—POLY-Y—S—W, where POLY, Y and S—W are asdefined above. Typically, though not necessarily, the polymeric reagentis symmetrical. An exemplary polymeric reagent of this structure,depicted below, is one in which POLY is polyethylene glycol (PEG), eachY is —(CH₂)₄—, and each —S—W is ortho-pyridyl disulfide (OPSS), asdepicted below, or SH. The PEG preferably has a molecular weight in therange of about 1000 to 5000 Da, e.g. 2000 or 3400 Da.

Further exemplary polymeric reagents include reagents of the generalformula PEG-[Y—S—W], where x is 1 or 2, Y is —(CH₂CH₂CH₂CH(CH₃))—, andS—W is SH or ortho-pyridyl disulfide (OPSS). When x is I, PEG ispreferably methoxy-terminated polyethylene glycol (mPEG). Such reagentsare depicted below (“Me” represents methyl here and elsewhere):

Further exemplary polymeric reagents include those of the generalformula PEG-[Y—S—W]_(x) where x is 1 or 2, Y is —(CH₂CH₂CH(CH₃))—, andS—W is SH or ortho-pyridyl disulfide (OPSS). When x is 1, PEG ispreferably mPEG. Such reagents are depicted below:

Another exemplary class of polymeric reagents is that of the generalformula PEG-[Y—S—W]_(x) where x is 1, Y is —(CH₂)₄—, —S—W is SH orortho-pyridyl disulfide (OPSS), and PEG is terminated with thestructure:

Such reagents are depicted generally below:

Preferably, the PEG attached to Y—SW has a molecular weight of about 500Da or less, or about 200 Da or less, e.g. where m=2 to 10, preferably 2to 4. In one embodiment, m=4. Each mPEG in the terminal branchedstructure shown preferably has a molecular weight of about 5 KDa toabout 20 KDa; e.g. n=about 110 to about 450. Each mPEG may be, forexample, 5, 10, 15 or 20 KDa in molecular weight.

The reagents described herein are characterized as being “linkerless”thiols; that is, where the water-soluble polymer is directly linked tothe hydrocarbon-based spacer group Y. For example, the oxygen atom of arepeating alkylene glycol unit of a poly(alkylene)glycol, such as—CH₂CH₂O— in PEG, is directly linked to Y. The absence of additionalheteroatoms, particularly in linkages such as esters, carbamates, oramides, between the active conjugating functionality, i.e., the thiol orprotected thiol, and the polymer reduces the potential for degradationof the conjugated polymer. Moreover, the presence of suchheteroatom-containing linkages, such as amides, in such reagents, cantrigger a deleterious immune response. This potential is eliminated orgreatly reduced by the current “linkerless” reagents.

In the polymeric reagents, the polymer segment POLY preferably has amolecular weight of at least 500 Da, particularly in embodiments wherePOLY is PEG. Various preferred embodiments of the polymer segment POLYare described in detail below. Preferably, the polymer segment “POLY” isa polyalkylene glycol, such as a polyethylene glycol (PEG). Thepolyethylene glycol may have various molecular weights, from about 88 toabout 100,000 Daltons, within the stipulations above. In selectedembodiments, the polyethylene glycol has a weight average molecular massfrom 148 (e.g. a trimer plus linking oxygen atom) to about 200 to about40,000 Daltons. Representative molecular weights include, for example,500, 1000, 2000, 3000, 3500, 5000, 7500, 10000, 15000, 20000, 25000,30000, and 40000 Daltons. Generally, difunctional or polyfunctionalreagents will employ POLY or PEG segments of lower molecular weight thanmonofunctional reagents.

The polymer can have a structure selected from the group consisting oflinear, branched, forked, multi-armed, and combinations thereof, asdescribed further herein.

The Polymer Segment, POLY

Representative water soluble polymers for use in preparing the polymericreagents of the invention include poly(alkylene glycols) such aspoly(ethylene glycol), poly(propylene glycol) (“PPG”), copolymers ofethylene glycol and propylene glycol, poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazoline, andpoly(N-acryloylmorpholine). POLY can be a homopolymer, an alternatingcopolymer, a random copolymer, a block copolymer, an alternatingtripolymer, a random tripolymer, or a block tripolymer of any of theabove. The water-soluble polymer segment is preferably a poly(ethyleneglycol) (“PEG”) or a derivative thereof.

Preferably, the polymer is a hydrophilic polymer; i.e., a polymercontaining fewer than about 25 subunits of propylene oxide or othersimilar hydrophobic polymer segments. The polymer may, in an alternativeembodiment, have no propylene oxide or similar hydrophobic subunits. Inone instance, the polymer is preferably not a pluronic-type polymer. Inyet another particular embodiment, the polymer is preferably not boundto a solid phase support.

The polymer segment can have any of a number of different geometries,for example, POLY can be linear, branched, or multiarmed. Mosttypically, POLY is linear or is branched, for example, having 2 polymerarms. Although much of the discussion herein is focused upon PEG as anillustrative POLY, the discussion and structures presented herein can bereadily extended to encompass any of the water-soluble polymer segmentsdescribed above.

Although water-soluble polymers bearing only one or two thiolfunctionalities are typically used and illustrated herein, polymersbearing two, three, four, five, six, seven, eight, nine, ten, eleven,twelve or more such functionalities can be used. Non-limiting examplesof the upper limit of the number of thiol moieties associated with thewater-soluble polymer segment include from about 1 to about 500, from 1to about 100, from about 1 to about 80, from about 1 to about 40, fromabout 1 to about 20, and from about 1 to about 10.

A preferred type of water soluble polymer, PEG, encompassespoly(ethylene glycol) in any of its linear, branched or multi-arm forms,including end-capped PEG, forked PEG, branched PEG, pendant PEG, and PEGcontaining one or more degradable linkages separating the monomersubunits, to be more fully described below. The number of repeatingethylene glycol units in a PEG polymer segment typically ranges fromabout 3 to about 4,000, or from about 12 to about 3,000, or morepreferably from about 20 to about 1,000.

Preferred end-capped PEGs are those having as an end-capping moiety suchas alkoxy, substituted alkoxy, alkenyloxy, substituted alkenyloxy,alkynyloxy, substituted alkynyloxy, aryloxy, substituted aryloxy.Preferred end-capping groups are C₁-C₂₀ alkoxy such as methoxy, ethoxy,and benzyloxy. The end-capping group can also advantageously comprise aphospholipid. Exemplary phospholipids include phosphatidylcholines, suchas dilauroylphosphatidylcholine, dioleyl phosphatidylcholine,dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine,behenoyl phosphatidylcholine, arachidoyl phosphatidylcholine, andlecithin.

A terminus of the polymer which is not thiolated may include, as analternative to a capping group, a reactive moiety, which is preferablyin protected form. Examples of such reactive moieties include hydroxy,amino, ester, carbonate, aldehyde, acetal, aldehyde hydrate, ketone,ketal, ketone hydrate, alkenyl, acrylate, methacrylate, acrylamide,sulfone, carboxylic acid, isocyanate, isothiocyanate, hydrazide, urea,maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide,alkoxy, benzyloxy, silane, lipid, phospholipid, biotin, and fluorescein,including activated and protected forms thereof where applicable.Preferred are functional groups such as N-hydroxysuccinimidyl ester,1-hydroxybenzotriazolyl carbonate, amine, vinylsulfone, maleimide,N-succinimidyl carbonate, hydrazide, succinimidyl propionate,succinimidyl butanoate, succinimidyl succinate, succinimidyl ester,glycidyl ether, oxycarbonylimidazole, p-nitrophenyl carbonate, aldehyde,orthopyridyl-disulfide, and acrylol.

These and other functional groups are described, for example, in thefollowing references, all of which are incorporated by reference herein:N-succinimidyl carbonate (U.S. Pat. Nos. 5,281,698 and 5,468,478), amine(Buckmann et al., Makromol. Chem. 182:1379 (1981); Zalipsky et al., Eur.Polym. J. 19:1177 (1983)), hydrazide (Andresz et al., Makromol. Chem.179:301 (1978)), succinimidyl propionate and succinimidyl butanoate(Olson et al., in Poly(ethylene glycol): Chemistry & BiologicalApplications, pp. 170-181, Harris & Zalipsky, Eds., ACS, Washington,D.C. (1997); U.S. Pat. No. 5,672,662), succinimidyl succinate(Abuchowski et al., Cancer Biochem. Biophys. 7:175 (1984) and Joppich etal., Makromol. Chem. 180:1381 (1979)), succinimidyl ester (U.S. Pat. No.4,670,417), benzotriazole carbonate (U.S. Pat. No. 5,650,234), glycidylether (Pitha et al., Eur. J. Biochem. 94:11 (1979); Elling et al.,Biotech. Appl. Biochem. 13:354 (1991)), oxycarbonylimidazole (Beauchampet al., Anal. Biochem. 131:25 (1983); Tondelli et al., J. ControlledRelease 1:251 (1985)), p-nitrophenyl carbonate (Veronese et al., Appl.Biochem. Biotech. 11:141 (1985); Sartore et al., Appl. Biochem. Biotech.27:45 (1991)), aldehyde (Harris et al., J. Polym. Sci. Chem. Ed. 22:341(1984); U.S. Pat. No. 5,824,784; U.S. Pat. No. 5,252,714), maleimide(Goodson et al., Bio/Technology 8:343 (1990); Romani et al., inChemistry of Peptides and Proteins 2:29 (1984); Kogan, Synthetic Comm.22:2417 (1992)), orthopyridyl-disulfide (Woghiren et al., Bioconj. Chem.4:314 (1993)), acrylol (Sawhney et al., Macromolecules 26:581 (1993)),and vinylsulfone (U.S. Pat. No. 5,900,461).

The POLY types described encompass linear polymer segments as well asbranched or multi-arm polymer segments. Examples include PEG moleculeshaving 2 arms, 3 arms, 4 arms, 5 arms, 6 arms, 7 arms, 8 arms or more.Branched polymers used to prepare the thiol polymers of the inventionmay possess anywhere from 2 to 300 or so reactive termini. Preferred arebranched or multi-arm PEGs having 2-8 polymer arms. Branched or multiarmpolymers for use in preparing a polymeric thiol of the invention includethose represented more generally by the formula R(POLY)_(n), where R isa central or core molecule from which extends 2 or more POLY arms suchas PEG. The variable n represents the number of POLY arms, where each ofthe polymer arms can independently be end-capped or possess a hydroxylor other reactive group at its terminus, where at least one polymer armpossesses such a reactive group. Branched PEGs such as those representedgenerally by the formula, R(PEG)_(n), above possess at least 2 polymerarms, up to about 300 polymer arms (i.e., n ranges from 2 to about 300).Preferably, such branched PEGs possess from 2 to about 25 polymer arms,more preferably from 2 to about 20 polymer arms, and even morepreferably from 2 to about 15 polymer arms or fewer. Most preferred aremulti-armed polymers having 3, 4, 5, 6, 7 or 8 arms.

Preferred core molecules in branched PEGs as described above arepolyols. Such polyols include aliphatic polyols having from 1 to 10carbon atoms and from 1 to 10 hydroxyl groups, including ethyleneglycol, alkane diols, alkyl glycols, alkylidene alkyl diols, alkylcycloalkane diols, 1,5-decalindiol,4,8-bis(hydroxymethyl)tricyclodecane, cycloalkylidene diols,dihydroxyalkanes, trihydroxyalkanes, tetrahydroxyalkanes and the like.Also, ethers of some or all of the former class may serve as coremolecules, including dipentaerythritol, tripentaerthritol, hexaglyceroland the like. Cycloaliphatic polyols may also be employed, includingstraight chained or closed-ring sugars and sugar alcohols, such asmannitol, sorbitol, inositol, xylitol, quebrachitol, threitol, arabitol,erythritol, adonitol, ducitol, facose, ribose, arabinose, xylose,lyxose, rhamnose, galactose, glucose, fructose, sorbose, mannose,pyranose, altrose, talose, tagitose, pyranosides, sucrose, lactose,maltose, and the like. Additional aliphatic polyols include derivativesof glyceraldehyde, glucose, ribose, mannose, galactose, and relatedstereoisomers. Other core polyols that may be used include crown ether,cyclodextrins, dextrins and other carbohydrates such as starches andamylose. Preferred polyols include glycerol, pentaerythritol, sorbitol,and trimethylolpropane.

A multi-arm structure corresponding to a polymeric thiol of theinvention can be represented by R—(POLY-Y—S—W)_(x), where POLY, Y, andS—W are as defined above, R represents the core molecule of the multiarmstructure, and x is preferably 3 to about 8. Each of the polymer armscan independently be end-capped or possess a thiol group at itsterminus, where at least one polymer arm possesses a thiol (or protectedthiol) group. Multi-arm PEGs suitable for preparing such structures areavailable from Nektar Therapeutics (Huntsville, Ala.).

Alternatively, the polymer segment may possess an overall forkedstructure, e.g., of the type PEG-(Y—S—W)₂. This type of polymer segmentis useful for reaction with two active agents, where the two activeagents are positioned in a precise or predetermined distance apart,depending upon the selection of Y.

Representative PEGs having either linear or branched structures for usein preparing the conjugates of the invention may be purchased fromNektar Therapeutics (Huntsville, Ala.). Illustrative structures aredescribed in Nektar's 2004 catalogue entitled “Polyethylene Glycol andDerivatives for Advanced PEGylation,” the contents of which areexpressly incorporated herein by reference.

In any of the representative structures provided herein, one or moredegradable linkages may be contained in the POLY segment, to allowgeneration in vivo of a conjugate having a smaller POLY chain than inthe initially administered conjugate. Appropriate physiologicallycleavable linkages include but are not limited to ester, carbonateester, carbamate, sulfate, phosphate, acyloxyalkyl ether, acetal, andketal. Such linkages will preferably be stable upon storage and uponinitial administration.

The molecular weight of POLY typically falls in one or more of thefollowing ranges: about 100 to about 100,000 Daltons; about 500 to about80,000 Daltons; about 1,000 to about 50,000 Daltons; about 2,000 toabout 25,000 Daltons; and about 5,000 to about 20,000 Daltons. Exemplarymolecular weights include about 1,000, about 5,000, about 10,000, about15,000, about 20,000, about 25,000, about 30,000, and about 40,000Daltons. Low molecular weight POLYs possess molecular weights of about250, 500, 750, 1000, 2000, or 5000 Daltons. Exemplary thiolated polymerscomprise PEGs having a molecular weight selected from the groupconsisting of 5,000, 20,000, and 40,000 Daltons.

In particular embodiments of the invention, a polymeric thiol reagent asprovided herein possesses a PEG segment having one of the followingmolecular weights: 500, 1000, 2000, 3000, 5000, 10,000, 15,000, 20,000,30,000 and 40,000 Daltons.

In terms of the number of subunits, PEGs for use in the invention willtypically comprise a number of (—OCH₂CH₂—) subunits falling within oneor more of the following ranges: from 12 to about 4000 subunits, fromabout 15 to about 2000 subunits, from about 20 to about 1000 subunits,from about 25 to about 750 subunits, and from about 30 to about 500subunits.

Preparation of Reagents

One method of preparing “linkerless” polyalkyleneoxy-thiol reagents isshown in Scheme I. In the reaction shown, a polyalkylene glycol having acapping group at one terminus, such as monomethoxy PEG, is alkoxylatedwith a strong base, such as NaH, and this reagent is combined with adi(haloalkylsulfide) to form a polymeric diether disulfide. Thisintermediate can then be cleaved with a reducing agent such asdithiothreitol (DTT) to give the polyalkyleneoxy-thiol reagent.

In another synthetic route, shown in Scheme 2, a di(hydroxyalkylsulfide)is used as a core material for polymerization of ethylene oxide, forminga PEG diether disulfide. The termini are capped with, for example,methyl groups. The disulfide can then be cleaved with a reducing agentsuch as dithiothreitol (DTT) to give the polyalkyleneoxy-thiol reagent.

A further strategy is illustrated in Scheme 3. In this route, a reagentPOLY-Y—OH, where Y is as defined above, is provided. Such reagents canbe prepared, for example, by reaction of a POLY-OH, such as m-PEG-OH,with a strong base such as NaH to form the alkoxide salt, followed byreaction with a haloalkanol, such as 4-bromo-1-butanol. The terminalhydroxy group is converted to a leaving group, such as tosylate ormesylate, and this compound is then reacted with thiourea, displacingthe leaving group. The terminal thiouronium salt is then cleaved withbase to give the terminal thiol. A variation on this scheme in which theleaving group is a halide is employed in Example 1 below.

In any of these reagents, the thiol can be protected using a thiolprotecting moiety such trityl, thioethers such as alkyl and benzylthioethers, including monothio, dithio and aminothio acetals,thioesters, thiocarbonates, thiocarbamates, and sulfenyl derivatives.The use of a protecting group during storage further reduces thetendency of the reagents to dimerize. The thiol may also be converted toan ortho-pyridyl disulfide (OPSS), as shown in Scheme 3, which is stableunder standard conditions of storage. Under appropriate reactionconditions, the OPSS group reacts smoothly with thiol groups in moietiesto be conjugated to the water-soluble polymer, as shown in Example 2.

Preferably, the polymeric reagents of the invention are stored under aninert atmosphere, such as argon or nitrogen. It is also preferable tominimize exposure of the polymers of the invention to moisture. Thus,preferred storage conditions are under dry argon or another dry inertgas at temperatures below about −15° C. Storage under low temperatureconditions is preferred, since rates of undesirable side reactions areslowed at lower temperatures. For example, when the polymer segment isPEG, the PEG can react slowly with oxygen to form peroxides, ultimatelyleading to chain cleavage and increasing the polydispersity of the PEGreagents. In view of the above, it is additionally preferred to storethe polymers of the invention in the dark.

Polymer Conjugates

The present invention also encompasses conjugates formed by reaction ofany of the herein described polymeric thiol reagents. In general, thepolymeric reagents of the invention are useful for conjugation to activeagents bearing at least one thiol group available for reaction.

A conjugate of the invention will typically have the structurePOLY-[Y—S—S-A]_(x), where POLY is as defined above, and in preferredembodiments is a polyethylene glycol (PEG); x is 1 to 25, and “A”represents the residue of the active agent following conjugation. Inselected embodiments, x is 1 to 8, 1 to 6, or 1 to 4; in furtherembodiments, x is 1 or x is 2. Y is a divalent linking group having atleast four carbon atoms and consisting of a saturated or unsaturatedhydrocarbon backbone which is three to ten, preferably three to eight,carbon atoms in length and has substituents which are independentlyselected from hydrogen, lower alkyl, lower alkenyl, and non-interferingsubstituents as defined herein, where two such alkyl and/or alkenylsubstituents on different carbon atoms of the backbone may be linked soas to form a cycloalkyl, cycloalkenyl, or aryl group.

In selected embodiments, Y has the structure —(CR¹R²)n—, where n is 3 to10, preferably 3 to 8, and each of R¹ and R² is independently selectedfrom hydrogen, lower alkyl, lower alkenyl, and a non-interferingsubstituent. Two groups R¹ and R² on different carbon atoms may belinked to form a cycloalkyl, cycloalkenyl, or aryl group. The sulfuratom of the thiol (or protected thiol) is attached to an sp³ hybridizedcarbon atom of Y, rather than to an aryl ring or double bond.

In selected embodiments of Y, Y is C₃-C₈ alkylene or a combination ofC₃-C₈ alkylene with C₅-C₈ cycloalkylene or aryl, any of which mayinclude one or more non-interfering substituents, as defined above.Preferably, zero to two, more preferably zero or one, suchnon-interfering substituents are included.

In further embodiments, Y is a linear or branched alkylene having theformula —(CR¹R²)n—, where n is 4 to 8, and each of R¹ and R² isindependently selected from hydrogen, lower alkyl, lower alkenyl, and anon-interfering substituent. Preferably, n is 4 to 6, and each of R¹ andR² is independently selected from hydrogen and methyl. In oneembodiment, each of R¹ and R² is hydrogen with respect to the niterations of —(CR¹R²)—. In another embodiment, each of R¹ and R² ishydrogen with the exception of R¹ on a carbon adjacent said sulfur atom(α-carbon), said R¹ being lower alkyl, preferably methyl or ethyl.

In embodiments of Y in which Y is —(CR¹R²)n—, where n is 4 to 8, and twogroups R¹ and R² on different carbon atoms are linked to form acycloalkyl, cycloalkenyl, or aryl group, the cycloalkyl group ispreferably a cyclopentyl or cyclohexyl group.

In another aspect, a conjugate of the invention can have the structurePOLY_(A)-L-SS—Y—POLY_(B)-Y′—SS-A. Each POLY is a water soluble polymersegment, as defined above, where POLY_(B) is of low molecular weight,e.g. 10 KDa or less, preferably 5 KDa or less, and more preferably 2 KDaor less, and the combined molecular weight of POLY_(A) and POLY_(B) isat least 3 KDa. The molecular weight of POLY_(A) is generally, thoughnot necessarily, of medium to high molecular weight, e.g. greater thanabout 2 KDa, preferably 5 KDa or greater, and more preferably 10 KDa orgreater.

Each of Y and Y′ is a spacer group, as defined above for Y, and they maybe the same or different. Generally, Y and Y′ are identical spacergroups. L is a linker between POLY_(A) and the adjacent disulfidelinkage. Typically, such a linker is a direct bond or a chain of atomsup to about ten atoms in length, containing groups preferably selectedfrom alkyl (C—C), alkenyl, ether, ester, amide, carbamate, and thioesterlinkages. L may be, but is not necessarily, an embodiment of Y asdescribed herein.

These conjugates are generally the product of a reaction sequence(illustrated in Examples 8-11 below) in which a low molecular weightreagent of the form W—S—S—Y—POLY_(B)-Y′—S—S—W, where S—W is a thiol or,preferably, a thiol-reactive derivative such as OPSS, is first reactedwith a biologically active molecule A-SH, e.g. a protein having (ormodified to have) a free cysteine residue, to form an intermediateA-S—S—Y—POLY_(B)-Y′—S—S—W. This intermediate is then reacted with a(typically) higher molecular weight reagent of the form POLY_(A)-L-S—S—Wto give the final conjugate.

An advantage of such a scheme, particularly for biological moleculeswith hindered thiol groups, is that a low molecular weight reagent isable to react more efficiently with such a hindered thiol group thanwould a higher molecular weight reagent. A higher molecular weightpolymer can thus be attached to A in greater yield via this scheme thanif it were reacted directly with the hindered thiol.

However, if the initial reagent W—S—S—Y—POLY_(B)-Y′—S—S—W lacks thehydrophilic spacer group(s) Y as described herein, the scheme may faildue to low yields in the attachment steps. This difference isillustrated in comparative Examples 9 and 10 below.

In general, the conjugates provided herein are preferably water solubleor dispersible, although the polymeric thiol reagents may also beconjugated to a solid support or surface having active thiol groups.

A thiol group, such as in a cysteine residue, for coupling to anactivated polymer of the invention may be naturally occurring (i.e.,occurring in the protein or other molecule in its native form), or itmay be introduced, e.g. by inserting into the native sequence of aprotein in place of a naturally-occurring amino acid, using standardgenetic engineering techniques.

When the active agent contains few or only one reactive thiol group(s),the resulting composition may advantageously contain only a singlepolymer conjugate species. This is useful in conjugation to proteins,which typically have a relatively low number of sulfhydryl groups (ascompared to other active groups such as amines) accessible forconjugation. Covalent attachment via thiol groups can thus result inmore selective modification of the target protein. Accordingly, the useof polymeric thiols can allow greater control over the resulting polymerconjugate, both in the number of polymer derivatives attached to theparent protein and the position of polymer attachment.

Candidate Molecules for Conjugation

A biologically active agent for use in preparing a conjugate of theinvention may fall into one of a number of structural classes, includingbut not limited to peptides, polypeptides, proteins, antibodies,polysaccharides, steroids, nucleotides, oligonucleotides,polynucleotides, fats, electrolytes, small molecules (preferablyinsoluble small molecules), and the like. Preferably, an active agentfor coupling to a polymer of the invention possesses a native sulfhydrylgroup or is modified to contain at least one reactive sulfhydryl groupsuitable for coupling.

Preferred peptides or proteins for coupling to a polymeric thiol of theinvention include EPO, IFN-α, IFN-β, IFN-γ, consensus IFN, Factor VII,Factor VIII, Factor IX, IL-2, Remicade™ (infliximab), Rituxan™(rituximab), Enbrel™ (etanercept), Synagis™ (palivizumab), Reopro™(abciximab), Herceptin™ (trastuzimab), tPA, Cerizyme™ (imiglucerase),hepatitis-B vaccine, rDNAse, alpha-1 proteinase inhibitor, G-CSF,GM-CSF, hGH, insulin, FSH, and PTH. In selected embodiments, the proteinis G-CSF or GM-CSF.

The above exemplary biologically active agents are meant to encompass,where applicable, analogues, agonists, antagonists, inhibitors, isomers,and pharmaceutically acceptable salt forms thereof. In reference topeptides and proteins, the invention is intended to encompass synthetic,recombinant, native, glycosylated, and non-glycosylated forms, as wellas biologically active fragments thereof. The above biologically activeproteins are additionally meant to encompass variants having one or moreamino acids substituted (e.g., cysteine), deleted, or the like, as longas the resulting variant protein possesses at least a certain degree ofactivity of the parent (native) protein.

Suitable agents may be selected from, for example, hypnotics andsedatives, psychic energizers, tranquilizers, respiratory drugs,anticonvulsants, muscle relaxants, antiparkinson agents (dopamineantagonists), analgesics, anti-inflammatories, antianxiety drugs(anxiolytics), appetite suppressants, antimigraine agents, musclecontractants, anti-infectives (antibiotics, antivirals, antifungals,vaccines) antiarthritics, antimalarials, antiemetics, anepileptics,bronchodilators, cytokines, growth factors, anti-cancer agents,antithrombotic agents, antihypertensives, cardiovascular drugs,antiarrhythmics, antioxidants, anti-asthma agents, hormonal agentsincluding contraceptives, sympathomimetics, diuretics, lipid regulatingagents, antiandrogenic agents, antiparasitics, anticoagulants,neoplastics, antineoplastics, hypoglycemics, nutritional agents andsupplements, growth supplements, antienteritis agents, vaccines,antibodies, diagnostic agents, and contrasting agents.

Other specific examples of active agents include but are not limited toasparaginase, amdoxovir (DAPD), antide, becaplermin, calcitonins,cyanovirin, denileukin diftitox, erythropoietin (EPO), EPO agonists(e.g., peptides from about 10-40 amino acids in length and comprising aparticular core sequence as described in WO 96/40749), dornase alpha,erythropoiesis stimulating protein (NESP), coagulation factors such asFactor V, Factor VII, Factor VIM, Factor VIII, Factor IX, Factor X,Factor XII, Factor XIII, von Willebrand factor; ceredase, cerezyme,alpha-glucosidase, collagen, cyclosporin, alpha defensins, betadefensins, exedin-4, granulocyte colony stimulating factor (GCSF),thrombopoietin (TPO), alpha-1 proteinase inhibitor, elcatonin,granulocyte macrophage colony stimulating factor (GMCSF), fibrinogen,filgrastim, growth hormones human growth hormone (hGH), growth hormonereleasing hormone (GHRH), GRO-beta, GRO-beta antibody, bone morphogenicproteins such as bone morphogenic protein-2, bone morphogenic protein-6,OP-1; acidic fibroblast growth factor, basic fibroblast growth factor,CD-40 ligand, heparin, human serum albumin, low molecular weight heparin(LMWH), interferons such as interferon alpha, interferon beta,interferon gamma, interferon omega, interferon tau, consensusinterferon; interleukins and interleukin receptors such as interleukin-1receptor, interleukin-2, interleukin-2 fusion proteins, interleukin-1receptor antagonist, interleukin-3, interleukin-4, interleukin-4receptor, interleukin-6, interleukin-8, interleukin-12, interleukin-13receptor, interleukin-17 receptor; lactoferrin and lactoferrinfragments, luteinizing hormone releasing hormone (LHRH), insulin,pro-insulin, insulin analogues (e.g., mono-acylated insulin as describedin U.S. Pat. No. 5,922,675), amylin, C-peptide, somatostatin,somatostatin analogs including octreotide, vasopressin, folliclestimulating hormone (FSH), influenza vaccine, insulin-like growth factor(IGF), insulintropin, macrophage colony stimulating factor (M-CSF),plasminogen activators such as alteplase, urokinase, reteplase,streptokinase, pamiteplase, lanoteplase, and teneteplase; nerve growthfactor (NGF), osteoprotegerin, platelet-derived growth factor, tissuegrowth factors, transforming growth factor-1, vascular endothelialgrowth factor, leukemia inhibiting factor, keratinocyte growth factor(KGF), glial growth factor (GGF), T Cell receptors, CDmolecules/antigens, tumor necrosis factor (TNF), monocytechemoattractant protein-1, endothelial growth factors, parathyroidhoxuione (PTH), glucagon-like peptide, somatotropin, thymosin alpha 1,thymosin alpha 1 IIb/IIIa inhibitor, thymosin beta 10, thymosin beta 9,thymosin beta 4, alpha-1 antitrypsin, phosphodiesterase (PDE) compounds,VLA-4 (very late antigen-4), VLA-4 inhibitors, bisphosphonates,respiratory syncytial virus antibody, cystic fibrosis transmembraneregulator (CFTR) gene, deoxyreibonuclease (Dnase),bactericidal/permeability increasing protein (BPI), and anti-CMVantibody. Exemplary monoclonal antibodies include etanercept (a dimericfusion protein consisting of the extracellular ligand-binding portion ofthe human 75 kD TNF receptor linked to the Fc portion of IgG1),abciximab, afeliomomab, basiliximab, daclizumab, infliximab, ibritumomabtiuexetan, mitumomab, muromonab-CD3, iodine 131 tositumomab conjugate,olizumab, rituximab, and trastuzumab (herceptin).

Additional agents suitable for covalent attachment to a polymer includebut are not limited to amifostine, amiodarone, aminocaproic acid,aminohippurate sodium, aminoglutethimide, aminolevulinic acid,aminosalicylic acid, amsacrine, anagrelide, anastrozole, asparaginase,anthracyclines, bexarotene, bicalutamide, bleomycin, buserelin,busulfan, cabergoline, capecitabine, carboplatin, carmustine,chlorambucin, cilastatin sodium, cisplatin, cladribine, clodronate,cyclophosphamide, cyproterone, cytarabine, camptothecins, 13-cisretinoic acid, all trans retinoic acid; dacarbazine, dactinomycin,daunorubicin, deferoxamine, dexamethasone, diclofenac,diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estramustine,etoposide, exemestane, fexofenadine, fludarabine, fludrocortisone,fluorouracil, fluoxymesterone, flutamide, gemcitabine, epinephrine,L-Dopa, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan,itraconazole, goserelin, letrozole, leucovorin, levamisole, lisinopril,lovothyroxine sodium, lomustine, mechlorethamine, medroxyprogesterone,megestrol, melphalan, mercaptopurine, metaraminol bitartrate,methotrexate, metoclopramide, mexiletine, mitomycin, mitotane,mitoxantrone, naloxone, nicotine, nilutamide, octreotide, oxaliplatin,pamidronate, pentostatin, pilcamycin, porfimer, prednisone,procarbazine, prochlorperazine, ondansetron, raltitrexed, sirolimus,streptozocin, tacrolimus, tamoxifen, temozolomide, teniposide,testosterone, tetrahydrocannabinol, thalidomide, thioguanine, thiotepa,topotecan, tretinoin, valrubicin, vinblastine, vincristine, vindesine,vinorelbine, dolasetron, granisetron; formoterol, fluticasone,leuprolide, midazolam, alprazolam, amphotericin B, podophylotoxins,nucleoside antivirals, aroyl hydrazones, sumatriptan; macrolides such aserythromycin, oleandomycin, troleandomycin, roxithromycin,clarithromycin, davercin, azithromycin, flurithromycin, dirithromycin,josamycin, spiromycin, midecamycin, leucomycin, miocamycin, rokitamycin,andazithromycin, and swinolide A; fluoroquinolones such asciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin,moxifloxicin, norfloxacin, enoxacin, grepafloxacin, gatifloxacin,lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin,fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin,clinafloxacin, and sitafloxacin; aminoglycosides such as gentamicin,netilmicin, paramecin, tobramycin, amikacin, kanamycin, neomycin, andstreptomycin, vancomycin, teicoplanin, rampolanin, mideplanin, colistin,daptomycin, gramicidin, colistimethate; polymixins such as polymixin B,capreomycin, bacitracin, penems; penicillins includingpenicllinase-sensitive agents like penicillin G, penicillin V;penicllinase-resistant agents like methicillin, oxacillin, cloxacillin,dicloxacillin, floxacillin, nafcillin; gram negative microorganismactive agents like ampicillin, amoxicillin, and hetacillin, cillin, andgalampicillin; antipseudomonal penicillins like carbenicillin,ticarcillin, azlocillin, mezlocillin, and piperacillin; cephalosporinslike cefpodoxime, cefprozil, ceftbuten, ceftizoxime, ceftriaxone,cephalothin, cephapirin, cephalexin, cephradrine, cefoxitin,cefamandole, cefazolin, cephaloridine, cefaclor, cefadroxil,cephaloglycin, cefuroxime, ceforanide, cefotaxime, cefatrizine,cephacetrile, cefepime, cefixime, cefonicid, cefoperazone, cefotetan,cefmetazole, ceftazidime, loracarbef, and moxalactam, monobactams likeaztreonam; and carbapenems such as imipenem, meropenem, pentamidineisethiouate, albuterol sulfate, lidocaine, metaproterenol sulfate,beclomethasone diprepionate, triamcinolone acetamide, budesonideacetonide, fluticasone, ipratropium bromide, flunisolide, cromolynsodium, and ergotamine tartrate; taxanes such as paclitaxel; SN-38, andtyrphostines.

Methods of Conjugation

Suitable conjugation conditions are those conditions of time,temperature, pH, reagent concentration, solvent, and the like sufficientto effect conjugation between a polymeric thiol reagent and an activeagent. As is known in the art, the specific conditions depend upon,among other things, the active agent, the type of conjugation desired,the presence of other materials in the reaction mixture, and so forth.Sufficient conditions for effecting conjugation in any particular casecan be determined by one of ordinary skill in the art upon a reading ofthe disclosure herein, reference to the relevant literature, and/orthrough routine experimentation.

Exemplary conjugation conditions include carrying out the conjugationreaction at a pH of from about 6 to about 10, and at, for example, a pHof about 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10. The reaction isallowed to proceed from about 5 minutes to about 72 hours, preferablyfrom about 30 minutes to about 48 hours, and more preferably from about4 hours to about 24 hours or less. Temperatures for conjugationreactions are typically, although not necessarily, in the range of fromabout 0° C. to about 40° C.; conjugation is often carried out at roomtemperature or less. Conjugation reactions are often carried out in abuffer such as a phosphate or acetate buffer or similar system.

With respect to reagent concentration, an excess of the polymericreagent is typically combined with the active agent. In some cases,however, it is preferred to have stoichiometric amounts of the number ofreactive groups on the polymeric reagent to the amount of active agent.Exemplary ratios of polymeric reagent to active agent include molarratios of about 1:1 (polymeric reagent:active agent), 1.5:1, 2:1, 3:1,4:1, 5:1, 6:1, 8:1, or 10:1. The conjugation reaction is allowed toproceed until substantially no further conjugation occurs, which cangenerally be determined by monitoring the progress of the reaction overtime.

Progress of the reaction can be monitored by withdrawing aliquots fromthe reaction mixture at various time points and analyzing the reactionmixture by SDS-PAGE or MALDI-TOF mass spectrometry or any other suitableanalytical method. Once a plateau is reached with respect to the amountof conjugate formed or the amount of unconjugated polymer remaining, thereaction is assumed to be complete. Typically, the conjugation reactiontakes anywhere from minutes to several hours (e.g., from 5 minutes to 24hours or more). The resulting product mixture is preferably, but notnecessarily purified, to separate out excess reagents, unconjugatedreactants (e.g., active agent) undesired multi-conjugated species, andfree or unreacted polymer. The resulting conjugates can then be furthercharacterized using analytical methods such as MALDI, capillaryelectrophoresis, gel electrophoresis, and/or chromatography.

More preferably, a polymeric thiol of the invention is typicallyconjugated to a sulfhydryl-containing active agent at a pH of about 6-9(e.g., at 6, 6.5, 7, 7.5, 8, 8.5, or 9), more preferably at a pH ofabout 7-9, and even more preferably at a pH of about 7 to 8. Generally,a slight molar excess of polymeric reagent is employed, for example, a1.5 to 15-fold molar excess, preferably a 2-fold to 10 fold molarexcess. Reaction times generally range from about 15 minutes to severalhours, e.g., 8 or more hours, at room temperature. For stericallyhindered sulfhydryl groups, required reaction times may be significantlylonger.

Purification of Conjugates

Optionally, conjugates produced by reacting a polymeric thiol of theinvention with a biologically active agent are purified toobtain/isolate different species, e.g., PEG-species, or to removeundesirable reaction side-products.

If desired, PEG conjugates having different molecular weights can beisolated using gel filtration chromatography. While this approach can beused to separate PEG conjugates having different molecular weights, thisapproach is generally ineffective for separating positional isomershaving different PEGylation sites within a protein. For example, gelfiltration chromatography can be used to separate from each othermixtures of PEG 1-mers, 2-mers, 3-mers, etc., although each of therecovered PEG-mer compositions may contain PEGs attached to differentreactive groups within the protein.

Gel filtration columns suitable for carrying out this type of separationinclude Superdex™ and Sephadex™ columns available from AmershamBiosciences. Selection of a particular column will depend upon thedesired fractionation range desired. Elution is generally carried outusing a non-amine based buffer, such as phosphate, acetate, or the like.The collected fractions may be analyzed by a number of differentmethods, for example, (i) OD at 280 nm for protein content, (ii) BSAprotein analysis, (iii) iodine testing for PEG content (Sims, G. E. C.et al., Anal. Biochem, 107, 60-63, 1980), or alternatively, (iv) byrunning an SDS PAGE gel, followed by staining with barium iodide.

Separation of positional isomers can be carried out by reverse phasechromatography using, for example, an RP-HPLC C18 column (AmershamBiosciences or Vydac) or by ion exchange chromatography using an ionexchange column, e.g., a Sepharose™ ion exchange column available fromAmersham Biosciences. Either approach can be used to separatePEG-biomolecule isomers having the same molecular weight (positionalisomers).

Depending upon the intended use for the resulting PEG-conjugates,following conjugation, and optionally additional separation steps, theconjugate mixture may be concentrated, sterile filtered, and stored atlow temperatures from about −20° C. to about −80° C. Alternatively, theconjugate may be lyophilized, either with or without residual buffer andstored as a lyophilized powder. In some instances, it is preferable toexchange a buffer used for conjugation, such as sodium acetate, for avolatile buffer such as ammonium carbonate or ammonium acetate, that canbe readily removed during lyophilization, so that the lyophilizedprotein conjugate powder formulation is absent residual buffer.Alternatively, a buffer exchange step may be used using a formulationbuffer, so that the lyophilized conjugate is in a form suitable forreconstitution into a formulation buffer and ultimately foradministration to a mammal.

Pharmaceutical Compositions

The present invention also includes pharmaceutical preparationscomprising a conjugate as provided herein in combination with apharmaceutical excipient. Generally, the conjugate itself will be in asolid form (e.g., a precipitate or a lyphilizate) or in solution, whichcan be combined with a suitable pharmaceutical excipient that can be ineither solid or liquid form.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The preparation may also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfate, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant may be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines (althoughpreferably not in liposomal form), fatty acids and fatty esters;steroids, such as cholesterol; and chelating agents, such as EDTA, zincand other such suitable cations.

Acids or bases may be present as an excipient in the preparation.Nonlimiting examples of acids that can be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumerate, and combinationsthereof.

The pharmaceutical preparations encompass all types of formulations andin particular those that are suited for injection, e.g., powders thatcan be reconstituted as well as suspensions and solutions. The amount ofthe conjugate (i.e., the conjugate formed between the active agent andthe polymer described herein) in the composition will vary depending ona number of factors, but will optimally be a therapeutically effectivedose when the composition is stored in a unit dose container (e.g., avial). In addition, the pharmaceutical preparation can be housed in asyringe. A therapeutically effective dose can be determinedexperimentally by repeated administration of increasing amounts of theconjugate in order to determine which amount produces a clinicallydesired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, the excipient will be present in the composition inan amount of about 1% to about 99% by weight, preferably from about5%-98% by weight, more preferably from about 15-95% by weight of theexcipient, with concentrations less than 30% by weight most preferred.

These foregoing pharmaceutical excipients along with other excipientsare described in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), the “Physician's DeskReference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), andKibbe, A.H., Handbook of Pharmaceutical Excipients, 3^(rd) Edition,American Pharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical preparations of the present invention are typically,although not necessarily, administered via injection and are thereforegenerally liquid solutions or suspensions immediately prior toadministration. The pharmaceutical preparation can also take other formssuch as syrups, creams, ointments, tablets, powders, and the like. Othermodes of administration are also included, such as pulmonary, rectal,transdermal, transmucosal, oral, intrathecal, subcutaneous,intra-arterial, and so forth.

As previously described, the conjugates can be administered injectedparenterally by intravenous injection, or less preferably byintramuscular or by subcutaneous injection. Suitable formulation typesfor parenteral administration include ready-for-injection solutions, drypowders for combination with a solvent prior to use, suspensions readyfor injection, dry insoluble compositions for combination with a vehicleprior to use, and emulsions and liquid concentrates for dilution priorto administration, among others.

Methods of Administration

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with conjugate. The method comprisesadministering, generally via injection, a therapeutically effectiveamount of the conjugate (preferably provided as part of a pharmaceuticalpreparation). The method of administering may be used to treat anycondition that can be remedied or prevented by administration of theparticular conjugate. Those of ordinary skill in the art appreciatewhich conditions a specific conjugate can effectively treat. The actualdose to be administered will vary depend upon the age, weight, andgeneral condition of the subject as well as the severity of thecondition being treated, the judgment of the health care professional,and conjugate being administered. Therapeutically effective amounts areknown to those skilled in the art and/or are described in the pertinentreference texts and literature. Generally, a therapeutically effectiveamount will range from about 0.001 mg to 100 mg, preferably in dosesfrom 0.01 mg/day to 75 mg/day, and more preferably in doses from 0.10mg/day to 50 mg/day.

The unit dosage of any given conjugate (again, preferably provided aspart of a pharmaceutical preparation) can be administered in a varietyof dosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration five times a day, four times a day, threetimes a day, twice daily, once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

Cleavage of the water-soluble polymer portion of the conjugate in vivo,when desired, can be effected through the use of physiologicallycleavable and/or enzymatically degradable linkages, such as urethane,amide, carbonate or ester-containing linkages, in the polymer backbone.In this way, clearance of the conjugate (via cleavage of water-solublepolymer portions) can be modulated by selecting the polymer molecularsize and the type functional group that would provide the desiredclearance properties. One of ordinary skill in the art can determine theproper molecular size of the polymer as well as the cleavable linkages.For example, one of ordinary skill in the art, using routineexperimentation, can determine a proper molecular size and cleavablefunctional group by first preparing a variety of polymer derivativeswith different polymer weights and cleavable linkages, and thenobtaining the clearance profile (e.g., through periodic blood or urinesampling) by administering the polymer derivative to a patient andtaking periodic blood and/or urine sampling. Once a series of clearanceprofiles have been obtained for each tested conjugate, a suitableconjugate can be identified.

All articles, books, patents, patent publications and other publicationsreferenced herein are incorporated by reference in their entireties.

EXAMPLES

The following examples illustrate but in no way are intended to limitthe scope of the present invention. In one aspect, the Examplesillustrate the increased stability, during synthesis and conjugation, ofthe polymeric thiol reagents of the invention.

¹H NMR data was obtained using a 400 MHz spectrometer manufactured byBruker.

PEG reagents referred to are available from Nektar Therapeutics,Huntsville, Ala.

Example 1 Preparation of mPEG-(CH₂)₄-orthopyridyl disulfide(mPEG-4C-OPSS)

I. mPEG₅₀₀₀-butyl bromide

A solution of mPEG₅₀₀₀ (20.0 g, 0.004 mol) (NOF Corporation) in toluene(200 ml) was azeotropically dried by distilling off 50 ml toluene.Sodium hydride (0.8 g, 60% dispersion in mineral oil, 0.020 mol) wasadded, and the mixture was stirred for 1 h at 60° C. under an argonatmosphere. 1,4-Dibromobutane (9.0 g, 0.0417 mol) was added, and themixture was stirred overnight at 75° C. under argon. The mixture wasfiltered and concentrated under reduced pressure, and the residue wascombined with 850 ml cold ethyl ether. The precipitated product wasfiltered off and dried under reduced pressure. Yield 17.4 g. NMR(d₆-DMSO): 1.60 ppm (m, —O—CH₂—CH₂ —CH₂—CH₂—Br, 2H), 1.84 ppm (m,—O—CH₂—CH₂ —CH₂—Br, 2H), 3.24 ppm (s, —OCH₃, 3H), 3.51 ppm (s, PEGbackbone).

II. mPEG₅₀₀₀-butanethiol

To a solution of mPEG₅₀₀₀-butyl bromide (2.0 g, 0.0004 mol) in anhydrousethyl alcohol (20 ml), thiourea (0.31 g, 0.0041 mol) was added, and themixture was stirred overnight at 78° C. under argon. The solvent wasremoved by distillation under reduced pressure, and the residue wasdissolved in 1% aqueous NaOH (21 ml). This solution was heated for 2.5 hat 85° C. under argon. After cooling the solution to 35° C., the pH wasadjusted to 3 with 10% phosphoric acid. NaCl (6 g) was added, and theproduct was extracted with dichloromethane. The extract was dried withanhydrous sodium sulfate, and the product was precipitated with coldethyl ether. Yield 1.8 g. NMR (CDCl₃): 1.35 ppm (t, CH₂—SH, 1H), 1.69ppm (m, —O—CH₂—CH₂ —CH₂ —CH₂—SH, 4H), 2.55 ppm (m, —CH₂ —SH, 2H), 2.69ppm (t, —CH₂—S—S—CH₂ —, 4H, 1.2 mol %) 3.24 ppm (s, —OCH₃, 3H), 3.51 ppm(s, PEG backbone).

Iodometric analysis showed that the product contained 94% thiol groups.The NMR data, above, indicated that the product contained a very smallamount (1.2 mol % by NMR) of disulfide-linked dimer, formed by oxidationof thiol groups. No further purification of the thiol was required.

In contrast, the analogous preparation of mPEG₅₀₀₀-ethanethiol (i.e. thecorresponding reagent containing only a two-carbon spacer between thePEG and the thiol group) from mPEG₅₀₀₀-mesylate and thiourea, conductedin a similar manner, produced product containing about 15 mol % ofdisulfide-linked dimer containing dithiol group (see e.g. WO2004/063250). This level of dimer necessitates further purification oradditional chemical treatment to convert the dimer to the desiredPEG-thiol.)

III. mPEG₅₀₀₀-4C-OPSS

To a solution of mPEG₅₀₀₀-butanethiol (2.0 g, 0.0004 mol) in anhydrousmethyl alcohol (40 ml), 2,2′-dipyridyl disulfide (0.18 g, 0.00082 mol)was added, and the mixture was stirred for 4 h at room temperature underargon. The solvent was removed by distillation under reduced pressure,the residue was dissolved in dichloromethane (5 ml), and the product wasprecipitated with 50 ml of cold ethyl ether. Yield 1.7 g. NMR (CDCl₃):1.68 ppm (m, —O—CH₂—CH₂ —CH₂—CH₂—S—, 2H), 1.76 ppm (m, —O—CH₂—CH₂—CH₂—CH₂—S—, 2H), 2.82 ppm (t, CH₂ —S—, 2H), 3.38 ppm (s, —OCH₃, 3H), 3.52ppm (s, PEG backbone), 7.12, 7.68, 7.75, & 8.47 ppm (4 m, pyridylprotons, 4H).

Example 2 Preparation of PEG₂₀₀₀-di-((CH₂)₄-orthopyridyl disulfide)(PEG-di-(4C-OPSS), 2 KDa)

PEG₂₀₀₀-di-butyl bromide

A solution of PEG₂₀₀₀ (20.0 g, 0.020 equiv.) (NOF Corporation) intoluene (150 ml) was azeotropically dried by distilling off 50 mltoluene. Sodium hydride (6.0 g, 60% dispersion in mineral oil, 0.150mol) was added, and the mixture was stirred for 1 h at 60° C. under anargon atmosphere. 1,4-Dibromobutane (34.55 g, 1.600 mol) was added, andthe mixture was stirred overnight at 75° C. under argon. The mixture wasfiltered and concentrated under reduced pressure, and the residue wascombined with 850 ml cold ethyl ether. The precipitated product wasfiltered off and dried under reduced pressure. Yield 17.0 g. NMR(d₆-DMSO): 1.60 ppm (m, —O—CH₂—CH₂ —CH₂—CH₂—Br, 2H), 1.84 ppm (m,—O—CH₂—CH₂—CH₂ —CH₂—Br, 2H), 3.51 ppm (s, PEG backbone); substitution96.3%.

PEG₂₀₀₀-di(butanethiol)

To a solution of PEG₂₀₀₀-di(butyl bromide) (10.0 g, 0.0100 equiv.) inanhydrous ethyl alcohol (100 ml), thiourea (7.68 g, 99%, 0.100 mol) wasadded, and the mixture was stirred overnight at 78° C. under argon. Thesolvent was removed by distillation under reduced pressure, and theresidue was dissolved in 3.3% aqueous NaOH (180 ml). This solution washeated for 2.5 h at 85° C. under argon. After cooling the solution to35° C., 60 ml deionized water was added, and the pH was adjusted to 3with 10% phosphoric acid. The solution was washed with 50 ml ethylacetate, and the product was extracted with dichloromethane. The extractwas dried with anhydrous sodium sulfate and the solvent was removed bydistillation under reduced pressure. The crude product wasrecrystallised from isopropyl alcohol and dried under vacuum. Yield 7.8g. NMR (CDCl₃): 1.35 ppm (t, —CH₂—SH, 1H), 1.69 ppm (m, —O—CH₂—CH₂ —CH₂—CH₂—SH, 4H), 2.55 ppm (m, —CH₂ —SH, 2H), 2.69 ppm (t, —CH₂—S—S—CH₂ —,4H, 1.9 mol %), 3.64 ppm (s, PEG backbone).

The NMR data, above, indicated that the product contained a relativelysmall amount (1.9 mol % by NMR) of disulfide-linked dimer, formed byoxidation of thiol groups. No further purification of the thiol wasrequired.

In contrast, the analogous preparation of PEG₂₀₀₀-di-ethanethiol (i.e.the corresponding reagent containing only a two-carbon spacer betweenthe PEG and the thiol group) from PEG₂₀₀₀-di-mesylate and thiourea,conducted in a similar manner, produced product containing about 41 mol% of disulfide-linked dimer containing dithiol group.

PEG₂₀₀₀-di-(4C-OPSS)

To a solution of 2,2′-dipyridyl disulfide (0.89 g, 0.0040 mol) inanhydrous methyl alcohol (40 ml), PEG₂₀₀₀-di-butanethiol (2.0 g, 0.0020equiv.) was added, and the mixture was stirred for 3 h at roomtemperature under argon. The solvent was removed by distillation underreduced pressure, the residue was dissolved in dichloromethane (5 ml),and the product was precipitated with 50 ml of cold ethyl ether. Theprecipitation was repeated giving 1.0 g of white solid product. NMR(CDCl₃): 1.68 ppm (m, —O—CH₂—CH₂ —CH₂—CH₂—S—, 2H), 1.76 ppm (m,—O—CH₂—CH₂—CH₂ —CH₂—S—, 2H), 2.82 ppm (t, CH₂ , —S—, 2H), 3.64 ppm (s,PEG backbone), 7.12, 7.68, 7.75, & 8.47 ppm (4 m, pyridyl Hs, 4H).

Example 3 Preparation of mPEG_(10,000)-(CH₂)₄-orthopyridyl disulfide(mPEG-4C-OPSS, 10 KDa)

mPEG_(10,000)-butyl bromide

A solution of mPEG_(10,000) (20.0 g, 0.002 mol) (NOF Corporation) intoluene (200 ml) was azeotropically dried by distilling off 50 mltoluene. Sodium hydride (0.8 g, 60% dispersion in mineral oil, 0.0200mol) was added, and the mixture was stirred for 1 h at 60° C. under anargon atmosphere. 1,4-Dibromobutane (4.8 g, 0.0222 mol) was added, andthe mixture was stirred overnight at 75° C. under argon. The mixture wasfiltered and concentrated under reduced pressure, and the residue wascombined with 850 ml cold ethyl ether. The precipitated product wasfiltered off and dried under reduced pressure. Yield 18.5 g. NMR(d₆-DMSO): 1.60 ppm (m, —O—CH₂—CH₂ —CH₂—CH₂—Br, 2H), 1.84 ppm (m,—O—CH₂—CH₂—CH₂ , —CH₂—Br, 2H), 3.24 ppm (s, —OCH₃, 3H), 3.51 ppm (s, PEGbackbone); substitution 97.5%.

mPEG_(10,000)-butanethiol

To a solution of mPEG_(10,000)-butyl bromide (10.0 g, 0.0010 mol) inanhydrous ethyl alcohol (100 ml), thiourea (0.77 g, 99%, 0.0100 mol) wasadded, and the mixture was stirred overnight at 78° C. under argon. Thesolvent was removed by distillation under reduced pressure, and theresidue was dissolved in 1.0% aqueous NaOH (90 ml). This solution washeated for 3 h at 85° C. under argon. After cooling the solution to roomtemperature NaCl (10 g) was added and the pH was adjusted to 3 with 10%phosphoric acid. The product was extracted with dichloromethane. Theextract was dried with anhydrous sodium sulfate, and the solvent wasremoved by distillation under reduced pressure. The crude product wasdissolved in small amount of dichloromethane, precipitated with ethylether and dried under vacuum. Yield 9.0 g. NMR (CDCl₃): 1.35 ppm (t,CH₂—SH, 1H), 1.69 ppm (m, —O—CH₂—CH₂ —CH₂ —CH₂—SH, 4H), 2.55 ppm (m,—CH₂ —SH, 2H), 2.69 ppm (t, —CH₂—S—S—CH₂ —, 4H, 4.8 mol %), 3.38 ppm (s,—OCH₃, 3H), 3.64 ppm (s, PEG backbone). The NMR data, above, indicatedthat the product contained a relatively small amount (4.8 mol % by NMR)of disulfide-linked dimer, formed by oxidation of thiol groups. Nofurther purification of the thiol was required.

mPEG_(10,000)-4C-OPSS

To a solution of 2,2′-dipyridyl disulfide (0.10 g, 0.00045 mol) inanhydrous methyl alcohol (40 ml), mPEG_(10,000)-butanethiol (2.0 g,0.00020 equiv.) was added, and the mixture was stirred for 3 h at roomtemperature under argon. The solvent was removed by distillation underreduced pressure. The crude product was dissolved in dichloromethane (5ml) and precipitated with 50 ml of cold ethyl ether giving after drying1.8 g of white solid powder. NMR (CDCl₃): 1.68 ppm (m, —O—CH₂—CH₂—CH₂—CH₂—S—, 2H), 1.76 ppm (m, —O—CH₂—CH₂—CH₂ —CH₂—S—, 2H), 2.82 ppm (t,—CH₂ —S—, 2H), 3.38 ppm (s, —OCH₃, 3H), 3.64 ppm (s, PEG backbone),7.12, 7.68, 7.75, & 8.47 ppm (4 m, pyridyl protons, 4H).

Examples 4 and 5 illustrate the preparation of corresponding reagents ofhigher molecular weight.

Example 4 Preparation of mPEG_(20,000)-(CH₂)₄-orthopyridyl disulfide(mPEG-4C-OPSS, 20 KDa)

mPEG_(20,000)-butyl bromide

A solution of mPEG_(20,000) (20.0 g, 0.0010 mol) (NOF Corporation) intoluene (200 ml) was azeotropically dried by distilling off 50 mltoluene. Sodium hydride (0.4 g, 60% dispersion in mineral oil, 0.0100mol) was added, and the mixture was stirred for 1 h at 60° C. under anargon atmosphere. 1,4-Dibromobutane (2.4 g, 0.0111 mol) was added, andthe mixture was stirred overnight at 75° C. under argon. The mixture wasfiltered and concentrated under reduced pressure, and the residue wascombined with 850 ml cold ethyl ether. The precipitated product wasfiltered off and dried under reduced pressure. Yield 18.2 g. NMR(d₆-DMSO): 1.60 ppm (m, —O—CH₂—CH₂ —CH₂—CH₂—Br, 2H), 1.84 ppm (m,—O—CH₂—CH₂—CH₂ —CH₂—Br, 2H), 3.24 ppm (s, —OCH₃, 3H), 3.51 ppm (s, PEGbackbone); substitution 98.0%.

mPEG_(20,000)-butanethiol

To a solution of mPEG_(20,000)-butyl bromide (10.0 g, 0.5 mmol) inanhydrous ethyl alcohol (100 ml), thiourea (0.39 g, 99%, 0.0051 mol) wasadded, and the mixture was stirred overnight at 78° C. under argon. Thesolvent was removed by distillation under reduced pressure, and theresidue was dissolved in 1.0% aqueous NaOH (90 ml). This solution washeated for 3 h at 85° C. under argon. After cooling the solution to roomtemperature NaCl (10 g) was added, and the pH was adjusted to 3 with 10%phosphoric acid. The product was extracted with dichloromethane. Theextract was dried with anhydrous sodium sulfate, and the solvent wasremoved by distillation under reduced pressure. The crude product wasdissolved in a small amount of dichloromethane, precipitated with ethylether and dried under vacuum. Yield 8.2 g. NMR (CDCl₃): 1.35 ppm (t,—CH₂—SH, 1H), 1.69 ppm (m, —O—CH₂—CH₂ —CH₂ —CH₂—SH, 4H), 2.55 ppm (m,—CH₂ —SH, 2H), 2.69 ppm (t, —CH₂—S—S—CH₂ —, 4H, 3.4 mol %), 3.38 ppm (s,—OCH₃, 3H), 3.64 ppm (s, PEG backbone). The NMR data, above, indicatedthat the product contained a relatively small amount (3.4 mol % by NMR)of disulfide-linked dimer, formed by oxidation of thiol groups. Nofurther purification of the thiol was required.

mPEG_(20,000)-4C-OPSS

To a solution of 2,2′-dipyridyl disulfide (0.05 g, 0.00023 mol) inanhydrous methyl alcohol (40 ml), mPEG_(20,000)-butanethiol (2.0 g,0.00010 mol) was added, and the mixture was stirred for 3 h at roomtemperature under argon. The solvent was removed by distillation underreduced pressure, the residue was dissolved in dichloromethane (5 ml),and the product was precipitated with 50 ml ethyl ether giving 1.9 g ofwhite solid powder. NMR (CDCl₃): 1.68 ppm (m, —O—CH₂—CH₂ —CH₂—CH₂—S—,2H), 1.76 ppm (m, —O—CH₂—CH₂—CH₂ —CH₂—S—, 2H), 2.82 ppm (t, —CH₂ —S—,2H), 3.38 ppm (s, —OCH₃, 3H), 3.64 ppm (s, PEG backbone), 7.12, 7.68,7.75, & 8.47 ppm (4 m, pyridyl protons, 4H).

Example 5 Preparation of mPEG_(30,000)-(CH₂)₄-orthopyridyl disulfide(mPEG-4C-OPSS, 30 KDa)

mPEG_(30,000)-butyl bromide

A solution of mPEG_(30,000) (20.0 g, 0.00067 mol) (NOF Corporation) intoluene (150 ml) was azeotropically dried by distilling off 50 mltoluene. Sodium hydride (0.3 g, 60% dispersion in mineral oil, 0.00750mol) was added, and the mixture was stirred for 1 h at 60° C. under anargon atmosphere. 1,4-Dibromobutane (2.17 g, 0.0100 mmol) was added, andthe mixture was stirred overnight at 75° C. under argon. The mixture wasfiltered and concentrated under reduced pressure, and the residue wascombined with 850 ml cold ethyl ether. The precipitated product wasfiltered off and dried under reduced pressure. Yield 15.3 g. NMR(d₆-DMSO): 1.60 ppm (m, —O—CH₂—CH₂ —CH₂—CH₂—Br, 2H), 1.84 ppm (m,—O—CH₂—CH₂—CH₂ —CH₂—Br, 2H), 3.24 ppm (s, —OCH₃, 3H), 3.51 ppm (s, PEGbackbone); substitution 96.0%.

mPEG_(30,000)-butanethiol

To a solution of mPEG_(30,000)-butyl bromide (10.0 g, 0.00033 mol) inanhydrous ethyl alcohol (100 ml), thiourea (0.26 g, 99%, 0.00338 mol)was added, and the mixture was stirred overnight at 78° C. under argon.The solvent was removed by distillation under reduced pressure, and theresidue was dissolved in 1.0% aqueous NaOH (90 ml). This solution washeated for 2.5 h at 85° C. under argon. After cooling to roomtemperature NaCl (10 g) was added and the pH was adjusted to 3 with 10%phosphoric acid. The product was extracted with dichloromethane. Theextract was dried with anhydrous sodium sulfate and the solvent wasremoved by distillation under reduced pressure. The crude product wasdissolved in a small amount of dichloromethane, precipitated with ethylether and dried under vacuum. Yield 9.4 g. NMR (CDCl₃): 1.35 ppm (t,—CH₂—SH, 1H), 1.69 ppm (m, —O—CH₂—CH₂ —CH₂—CH₂—SH, 4H), 2.55 ppm (m,—CH₂ —SH, 2H), 2.69 ppm (t, —CH₂—S—S—CH₂ —, 4H, 3.8 mol %), 3.38 ppm (s,—OCH₃, 3H), 3.64 ppm (s, PEG backbone). The NMR data, above, indicatedthat the product contained a relatively small amount (3.8 mol % by NMR)of disulfide-linked dimer, formed by oxidation of thiol groups. Nofurther purification of the thiol was required.

mPEG_(30,000)-4C-OPSS

To a solution of 2,2′-dipyridyl disulfide (0.05 g, 0.00023 mol) inanhydrous methyl alcohol (60 ml), mPEG_(30,000)-butanethiol (3.0 g,0.00010 mol) was added, and the mixture was stirred for 3 h at roomtemperature under argon. The solvent was removed by distillation underreduced pressure, the residue was dissolved in dichloromethane (8 ml),and the product was precipitated with 60 ml ethyl ether giving 2.9 g ofwhite solid powder. NMR (CDCl₃): 1.68 ppm (m, —O—CH₂—CH₂ —CH₂—CH₂—S—,2H), 1.76 ppm (m, —O—CH₂—CH₂—CH₂ —CH₂—S—, 2H), 2.82 ppm (t, —CH₂ —S—,2H), 3.38 ppm (s, —OCH₃, 3H), 3.64 ppm (s, PEG backbone), 7.12, 7.68,7.75, & 8.47 ppm (4 m, pyridyl protons, 4H).

Example 6 Conjugation of BSA with mPEG₅₀₀₀-4C-OPSS and with mPEG₅₀₀₀-MAL(MAL=maleimide) (Comparative)

Reduction of BSA (Cleavage of Disulfide Linkages)

A 3.1 mg sample of BSA was added to a 5 mL ReactiVial™ containing 3.1 mL1×PBS pH 7.5. The solution was placed on a stir plate at medium speed. A4.62 mg sample of dithiothreitol (DTT) was added to the solution withstirring and allowed to react for 2 hrs at room temperature, reducingthe sample completely.

The reaction mixture was placed in a 350 mL Amicon StirCell with a10,000 MW PES membrane for removal of DTT. Buffer (1×PBS pH 7.5) wasadded to a volume of 350 mL, with stirring to prevent settling. Pressurewas applied to the apparatus (60 psi) until the volume was reduced to<10 mL. PBS was again added to a volume of 350 mL, and the process wasrepeated twice. A 1 mL aliquot was frozen for standards (gels, HPLC,etc.), and the remaining volume was used in the conjugation step.

Conjugation

Reduced BSA from step A (4 mL) was combined with 2.35 mg (10× excess)mPEG_(5K)-4C-OPSS, described in Example 1, in a 5 mL ReactiVial on astir plate set to the medium setting. A similar reaction mixture wasprepared using reduced BSA from step A (4 mL) and 2.35 mg (10× excess)mPEG_(5K)-MAL. (In mPEG-MAL, available from Nektar Therapeutics,Huntsville, Ala., maleimide is attached via the ring nitrogen to theterminal —OCH₂CH₂— of mPEG.) The vials were left at room temperature for60 hrs.

Analysis

The reaction mixtures were run on 10% Bis-Tris NuPAGE Gels (Invitrogen)using the following conditions.

4x LDS Sample Buffer (Invitrogen) 10 μL/sample Reaction Sample 30μL/sample 1x MES Running Buffer (Invitrogen) 600 mL MultiMark ProteinStandards (Invitrogen) 7 μL SimplyBlue Safe Stain (Invitrogen) 50 mLLoading Sample (a + b) 30 μL Voltage 200 V Amps 400 mA Time 36 min.

FIG. 1 shows the finished gel stained with SimplyBlue Safe Stain. FIG. 2shows the same gel additionally stained with Barium Iodine, fordetecting PEG.

The molecular weight and relative intensity of the species shown in thegel of FIG. 2 are also set forth in the table below:

Lane 2: Lane 3: Lane 4: Reduced PEG_(5K)-MAL PEG_(5K)-OPSS BSAconjugation reaction conjugation reaction MW Rel. Int. MW Rel. Int. MWRel. Int. 100718.7 0.0727 126546.9 0.0591 134703.1 0.0433 50521.2 0.927385120 0.0852 85120 0.0664 75000 0.1449 75460 0.0826 58440 0.3984 584400.4224 50077.5 0.1822 50077.5 0.2032 7883.1^(a) 0.1301 22090.9^(a)0.0806 6454.5^(a) 0.1015 ^(a)visualized by BaI (for detection of PEG)

The PEG_(5K)-MAL-BSA conjugation reaction yielded 39.8% mono PEGmers(58440 MW bands), and the mPEG_(5K)-4C-OPSS-BSA conjugation reactionyielded 42.2% mono PEGmers. Accordingly, the conjugation behavior of thepolymeric thiol reagent of the invention was better than that observedfor a reference polymeric reagent (maleimide-terminated polymer),indicating that significant dimerization of PEG-OPSS, which is typicalfor the corresponding reagent based on mPEG-ethanethiol, did not occur.

Example 7 PEGylation of Granulocyte-Colony Stimulating Factor (G-CSF)with mPEG_(10,000)-(CH₂)₄-orthopyridyl disulfide (mPEG_(10,000)-4C-OPSS)

A fifty-fold excess (relative to the amount of G-CSF in a measuredaliquot of stock G-CSF solution) of mPEG_(10,000)-(CH₂)₄-orthopyridyldisulfide (mPEG_(10,000)-4C-OPSS), as prepared in Example 3, wasdissolved in dimethylsulfoxide (DMSO) to form a 10% reagent solution.The 10% reagent solution was quickly added to the aliquot of stock G-CSFsolution (0.4 mg/ml in sodium phosphate buffer, pH 7.0) and mixed well.To allow for coupling of the mPEG-OPSS reagent to the free (i.e.,nonintraprotein-disulfide bond participating) cysteine residue atposition 17 of G-CSF, the reaction solution was placed on a RotoMix(Type 48200, Thermolyne, Dubuque IA) to facilitate conjugation at 37° C.After thirty minutes, another fifty-fold excess of mPEG_(10,000)-4C-OPSSwas added to the reaction solution, followed by mixing first for thirtyminutes at 37° C., and then for two hours at room temperature, tothereby form an mPEG_(10,000)-G-CSF conjugate solution.

The mPEG_(10,000)-G-CSF conjugate solution was characterized by SDS-PAGEand RP-HPLC. The PEGylation reaction was determined to yield 36% ofmPEG_(10,000)G-CSF conjugate (a monoPEGylated conjugate at a cysteineresidue of G-CSF). Cation-exchange chromatography was used to purify theconjugate.

The same approach can be used to prepare other conjugates usingmPEG-4C-OPSS reagents having other molecular weights.

Examples 8-10, following, employ an approach (illustrated schematicallybelow) in which a polymeric reagent having a relatively low molecularweight (PEG_(B) in the schematic) is initially attached to a moiety tobe conjugated (A), followed by attachment of a higher molecular weightpolymeric reagent (PEG_(A) in the schematic) to the polymeric portion ofthe conjugate formed from attachment of the low molecular weight reagentto the conjugated moiety. Using this approach, it is possible toefficiently modify a hindered site. In the Examples below, the hinderedsite is the partially buried free thiol-containing cysteine residue ofG-CSF.

Example 8a PEGylation of G-CSF with PEG₂₀₀₀-di-((CH₂)₄-orthopyridyldisulfide) and mPEG_(20,000)-butanethiol

In this Example, the bifunctional PEG-di-(4C-OPSS) reagent is insertedinto the sterically hindered free thiol via a disulfide linkage,followed by the coupling of mPEG_(20K)-butanethiol to the free residueof the PEG_(2,000)-di-(4C-OPSS) reagent, via a further disulfidelinkage.

PEG_(2,000)-di-(4C-OPSS), as prepared in Example 2, stored at −20° C.under argon, was warmed to ambient temperature. A fifty-fold excess(relative to the amount of G-CSF in a measured aliquot of stock G-CSFsolution) of the reagent was dissolved in DMSO to form a 10% solution.The 10% reagent solution was quickly added to the aliquot of stock G-CSFsolution (0.4 mg/ml in sodium phosphate buffer, pH 7.0) and mixed well.The reaction solution was placed on a RotoMix (Type 48200, Thermolyne,Dubuque Iowa), and was allowed to mix for one hour at 37° C., and thenfor two hours at room temperature. After the reaction was complete, thereaction solution was dialyzed against sodium phosphate buffer, pH 7.0,to remove excess free PEG_(2,000)-di-(4C-OPSS).

A fifty-fold excess (relative to G-CSF) of mPEG_(20,000)-butanethiol, asprepared in Example 4B, was then added to the dialyzed solution ofintermediate conjugate, followed by mixing for one hour at roomtemperature and then overnight at 4° C., to thereby form themPEG_(20,000)-PEG_(2,000)-GCSF conjugate. The product was characterizedby SDS-PAGE and RP-HPLC.

This approach can be used to prepare other conjugates, usingPEG-di-(4C-OPSS) and mPEG-4C-SH having other molecular weights, againwhere the PEG-di-(4C-OPSS) reagent is preferably of relatively lowmolecular weight.

Example 8b PEGylation of G-CSF with PEG₂₀₀₀-di-((CH₂)₄-orthopyridyldisulfide) and mPEG_(30,000)-butanethiol

The procedure of Example 8a was repeated using corresponding amounts ofPEG₂₀₀₀-di-((CH₂)₄-orthopyridyl disulfide) andmPEG_(30,000)-butanethiol, to obtain the correspondingmPEG_(30,000)-PEG_(2,000)-GCSF conjugate.

Other conjugates can be similarly prepared using PEG-di-(4C-OPSS) andmPEG-4C-SH having other molecular weights.

Examples 9-10 below differ from each other in that the low molecularweight PEG species of Example 9 contains a four-carbon hydrophiliclinker of the invention, while that of Example 10 contains only atwo-carbon linker. It can be seen that the linker of the inventionprovides significantly greater yields of conjugate.

Example 9 PEGylation of G-CSF with PEG₂₀₀₀-di-((CH₂)₄-orthopyridyldisulfide) and Branched PEG2_(40,000)-thiol

Again, these examples employ an approach involving initial attachment ofa polymeric reagent having a relatively small molecular weight (in thisExample, PEG_(2,000)-di-(4C-OPSS)) to a G-CSF moiety, followed byattachment of a relatively large molecular weight polymeric reagent (inthis Example, branched PEG2_(40,000)-thiol) to residue of thePEG_(2,000)-di-(4C-OPSS) reagent, through another disulfide linkage.

PEG_(2,000)-di-(4C-OPSS) as prepared in Example 2, stored at −20° C.under argon, was warmed to ambient temperature. A fifty-fold excess(relative to the amount of G-CSF in a measured aliquot of stock G-CSFsolution) of the warmed PEG_(2,000)-di-(4C-OPSS) was dissolved in DMSOto form a 10% reagent solution. The 10% reagent solution was quicklyadded to the aliquot of stock G-CSF solution (0.4 mg/ml in sodiumphosphate buffer, pH 7.0) and mixed well. The solution was placed on aRotoMix (Type 48200, Thermolyne, Dubuque Iowa) and allowed to mix forone hour at 37° C., then for two hours at room temperature. After thereaction was complete, the reaction solution was dialyzed against sodiumphosphate buffer, pH 7.0, to remove excess PEG2,000-di-(4C-OPSS).

A seventy five-fold excess (relative to G-CSF) of PEG_(40,000)-thiol(Nektar Therapeutics) was then added to the dialyzed conjugate solution,followed by mixing for three hours at room temperature and thenovernight at 4° C., to form a PEG2_(40,000)-PEG_(2,000)-G-CSF conjugate.The conjugate was characterized by SDS-PAGE and RP-HPLC. The final yieldof conjugate obtained was 35%.

Example 10 Comparative PEGylation Reaction of G-CSF withPEG₂₀₀₀di-((CH₂)-orthopyridyl disulfide) and id PEG2_(40,000)-thiol

The reaction procedure of Example 10 was essentially duplicated, using alow molecular weight PEG thiol reagent having a two-carbon rather than afour-carbon linker.

Accordingly, PEG_(2,000)-di-(2C-OPSS) from Nektar Therapeutics, storedat −20° C. under argon, was warmed to ambient temperature. A fifty-foldexcess (relative to the amount of G-CSF in a measured aliquot of stockG-CSF solution) of the reagent was dissolved in DMSO to form a 10%solution. This solution was quickly added to the aliquot of stock G-CSFsolution (0.4 mg/ml in sodium phosphate buffer, pH 7.0) and mixed well.The reaction solution was placed on a RotoMix (Type 48200, Thermolyne,Dubuque Iowa) and was allowed to mix for one hour at 37° C., then fortwo hours at room temperature. After the reaction was complete, thereaction solution was dialyzed against sodium phosphate buffer, pH 7.0,to remove excess PEG2,000-di-(2C-OPSS).

A seventy-fold excess (relative to G-CSF) of branchedPEG2_(40,000)-thiol (Nektar Therapeutics) was added to the dialyzedconjugate solution, followed by mixing for three hour at roomtemperature and overnight at 4° C. However, SDS-PAGE and RP-HPLCanalysis showed no detectable amount of the desiredPEG2_(40,000)-PEG_(2,000)-G-CSF conjugate.

Evidence suggests that the ethylene (C2)-linked PEG-OPSS reagentundergoes reductive cleavage to effectively destroy the reagent beforeit reacts with the target protein. The butylene (C4)-linked reagent ismore stable to such cleavage and thereby survives to give a much higheryield of conjugate.

Example 11 Preparation of mPEG₅₀₀₀-CH₂—CH₂—CH₂—CH(CH₃)-orthopyridyldisulfide (mPEG-(α-methyl)4C-OPSS, 5 KDa)

1-Methyl-4-bromo-1-butanol

2-Methyltetrahydrofuran (10.14 g, 0.0834 mol) was dissolved inchloroform (72 ml), and tetraethylammonium bromide (18.4 g, 0.0876 mol)was added. Boron trifluoride-diethyl etherate (11.12 ml, 0.0876 mol) wasthen added dropwise over 15 min, and the solution was stirred overnightat room temperature. The solution was cooled to 0-5° C. and washed withsaturated aqueous NaHCO₃ (80 ml). The organic layer was separated,washed with water (80 ml) and saturated aqueous NaCl (80 ml), and driedwith anhydrous Na₂SO₄. The solvent was removed by distillation, giving9.5 g of pale yellow viscous liquid.

NMR (d₆-DMSO): 1.04 ppm (d, —CH₃—, 3H), 1.43 ppm (b, —CH₂ —CH(CH₃)—OH,2H), 1.84 ppm (m, —CH₂ —CH₂—CH(CH₃)—OH, 2H), 3.53 ppm (t, —CH₂Br, 2H),3.60 ppm (m, —CH₂—CH(CH₃)—OH, 1H), 4.41 ppm (bs, —OH, 1H).

1-Bromo-4-methyl-4-benzyloxybutane

To a solution of 1-methyl-4-bromo-1-butanol (9.0 g, 0.05384 mol) andbenzyl 2,2,2-trichloroacetimidate (16.3 g,) in a mixture of anhydrouscyclohexane (100 ml) and anhydrous dichloromethane (50 ml) cooled to 0°C., trifluoromethanesulfonic acid (1.0 ml) was added, and the mixturewas stirred overnight at room temperature under argon. The mixture wasfiltered, washed with a saturated solution of NaHCO₃ (250 ml) anddeionized water (250 ml), and dried with anhydrous Na₂SO₄. The solventswere removed by distillation under reduced pressure. The crude product(9.2 g) was subjected to vacuum distillation, giving 7.2 g of colorlessviscous liquid.

NMR (d₆-DMSO): 1.14 ppm (d, —CH₃—, 3H), 1.57 ppm (m, —CH₂ —CH(CH₃)—OH,2H), 1.88 ppm (m, —CH₂ —CH₂—CH(CH₃)—OH, 2H), 3.53 ppm (bm, —CH₂Br, 2Hand —CH₂—CH(CH₃)—OH, 1H), 4.46 ppm (m, —CH₂-benzyl, 2H), 7.32 ppm (m,C₆H₅—, benzyl, 5H).

mPEG₅₀₀₀-4-methyl-4-benzyloxybutane

To an azeotropically dried solution of mPEG₅₀₀₀ (20.0 g, 0.004 mole)(NOF Corporation) in anhydrous toluene (200 ml), a 1.0M solution ofpotassium tert-butoxide in tert-butanol (16.0 ml, 0.0160 mole) and1-bromo-4-methyl-4-benzyloxybutane (3.10 g, 0.012 mole) were added. Thereaction mixture was stirred for 20 hours at 70° C. under nitrogen. Theresulting mixture was filtered and concentrated under vacuum to dryness.The crude product was dissolved in 30 ml of dichloromethane andprecipitated with 500 ml of isopropanol at 0-5° C. The final product wascollected through vacuum filtration and dried under vacuum overnight.Yield: 17.4 g.

NMR (d₆-DMSO): 1.14 ppm (d, —CH₃, 3H), 1.57 ppm (m, —CH₂ —CH(CH₃)—OH,2H), 1.88 ppm (m, —CH₂ —CH₂—CH(CH₃)—OH, 2H), 3.24 ppm (s, —OCH₃, 3H),3.51 ppm (s, polymer backbone), 4.46 ppm (m, —CH₂-benzyl, 2H), 7.32 ppm(m, C₆H₅—, benzyl, 5H).

mPEG₅₀₀₀-4-methyl-4-butanol

A mixture of mPEG₅₀₀₀-4-methyl-4-benzyloxybutane (15.0 g, 0.00300 mole),ethyl alcohol (150 ml), and palladium (10% on active carbon, 1.5 g) washydrogenated overnight under 45 psi of hydrogen. The mixture wasfiltered and the solvent was removed by distillation under reducedpressure. The crude product was dissolved in dichloromethane (25 ml) andprecipitated with 400 ml isopropyl alcohol at 0-5° C. The product wasfiltered off and dried under reduced pressure. Yield: 13.1 g.

NMR (d₆-DMSO): 1.14 ppm (d, —CH₃, 3H), 1.57 ppm (m, —CH₂ —CH(CH₃)—OH,2H), 1.88 ppm (m, —CH₂ —CH₂—CH(CH₃)—OH, 2H), 3.24 ppm (s, —OCH₃, 3H),3.51 ppm (s, polymer backbone), 4.45 ppm (bs, —OH, 1H).

mPEG₅₀₀₀-4-methyl-4-methanesulfonylbutane

A solution of mPEG₅₀₀₀-4-methyl-4-butanol (10.0 g, 0.0020 mole) intoluene (100 ml) was azeotropically dried by distilling off tolueneunder reduced pressure. The dried mPEG₅₀₀₀-4-methyl-4-butanol wasdissolved in a mixture of anhydrous toluene (100 ml) and anhydrousdichloromethane (20 ml). Triethylamine (0.9 ml, 0.0030 mole) andmethanesulfonyl chloride (0.45 ml, 0.0026 mole) were added, and themixture was stirred overnight at room temperature under nitrogen. Thesolvents were removed by distillation under reduced pressure. Theresidue was dissolved in dichloromethane (15 ml), and 250 ml isopropylalcohol was added. The precipitated product was filtered and dried undervacuum to yield 8.9 g of the white solid powder.

NMR (d₆-DMSO): 1.40 ppm (d, —CH₃, 3H), 1.57 ppm (m, —CH₂—CH(CH₃)—mesylate, 2H), 1.88 ppm (m, —CH₂ —CH₂—CH(CH₃)-mesylate, 2H),3.17 ppm (s, —CH₃, mesylate, 3H), 3.24 ppm (s, —OCH₃, 3H), 3.51 ppm (s,polymer backbone), 4.00 ppm (m, —CH-mesylate, 1H).

mPEG₅₀₀₀-4-methyl-4-butanethiol

To a solution of mPEG₅₀₀₀-4-methyl-4-methanesulfonylbutane (8.0 g,0.0016 mol) in anhydrous ethyl alcohol (80 ml), thiourea (1.24 g, 0.0163mol) was added, and the mixture was stirred overnight at 78° C. underargon. The solvent was removed by distillation under reduced pressure,and the residue was dissolved in 1% aqueous NaOH (84 ml). This solutionwas heated for 2.5 h at 85° C. under argon. After cooling the solutionto 35° C., the pH was adjusted to 3 with 10% phosphoric acid. NaCl (24g) was added, and the product was extracted with dichloromethane. Theextract was dried with anhydrous sodium sulfate, and the product wasprecipitated with cold ethyl ether. Yield 7.3 g.

NMR (CDCl₃): 1.24 ppm (d, —CH₃, 3H), 1.38 ppm (m, —CH₂ —CH(CH₃)—SH, 2H),1.54 ppm (d, CH—SH, 1H), 1.88 ppm (m, —CH₂ —CH₂—CH(CH₃)—SH, 2H), 2.83ppm (m, —CH₂—CH(CH₃)—SH, 1H), 2.05 ppm (m, —CH₂ —CH(CH₃)—S—S—CH(CH₃)—CH₂—, 4H, 0.7 mol %), 3.38 ppm (s, —OCH₃, 3H), 3.64 ppm (s, PEG backbone).

The NMR data, above, indicated that the product contained a very smallamount (0.7 mol % by NMR) of disulfide-linked dimer, formed by oxidationof thiol groups. No further purification of the thiol was required.

mPEG₅₀₀₀-CH₂—CH₂—CH₂—CH(CH₃)_(n)OPSS

To a solution of mPEG₅₀₀₀-4-methyl-4-butanethiol (2.0 g, 0.0004 mol) inanhydrous methyl alcohol (40 ml), 2,2′-dipyridyl disulfide (0.18 g,0.00082 mol) was added, and the mixture was stirred for 4 h at roomtemperature under argon. The solvent was removed by distillation underreduced pressure, the residue was dissolved in dichloromethane (5 ml),and the product was precipitated with 50 ml of cold ethyl ether. Yield1.7 g.

NMR (CDCl₃): 1.34 ppm (d, —CH₃, 3H), 1.68 ppm (m, —CH₂ —CH₂—CH(CH₃)—SH,2H), 1.88 ppm (m, —CH₂—CH₂ —CH(CH₃)—SH, 2H), 3.38 ppm (s, —OCH₃, 3H),3.64 ppm (s, PEG backbone), 7.12, 7.68, 7.75, & 8.47 ppm (4 in, pyridylprotons, 4H).

1. A polymer conjugate comprising the structure:POLY_(A)-L-S—S—Y-POLY_(B)-Y—S—S-A wherein: each of POLY_(A) and POLY_(B)is a water soluble polymer segment, where POLY_(B) has a molecularweight of 10 KDa or less, and the sum of the molecular weights ofPOLY_(A) and POLY_(B) is at least 3 KDa; each Y is independently adivalent linking group consisting of a saturated or unsaturatedhydrocarbon backbone which is three to ten carbon atoms in length andhas substituents which are independently selected from hydrogen, loweralkyl, lower alkenyl, and non-interfering substituents as definedherein, where two such alkyl and/or alkenyl substituents on differentcarbon atoms of the backbone may be linked so as to form a cycloalkyl,cycloalkenyl, or aryl group; L is a linker group; each S—S is adisulfide group attached to an sp³ hybridized carbon of the adjacent Y;and A is a covalently linked residue of a pharmacologically activemolecule.
 2. The conjugate of claim 1, wherein each Y is independently—(CR¹R²)n-, where n is 3 to 10, each of R¹ and R² is independentlyselected from hydrogen, alkyl, alkenyl, and a non-interferingsubstituent, and where two groups R¹ and R² on different carbon atomsmay be linked to form a cycloalkyl or aryl group.
 3. The conjugate ofclaim 2, where n is 3 to
 8. 4. The conjugate of claim 1, whereinPOLY_(A) has a molecular weight of at least 5 KDa.
 5. The conjugate ofclaim 2, wherein the two Y groups are identical.
 6. The conjugate ofclaim 2, wherein each Y is a linear or branched alkylene having theformula —(CR¹R²)n-, where n is 3 to 8, and each of R¹ and R² isindependently selected from hydrogen and lower alkyl.
 7. The conjugateof claim 1, wherein each of POLY_(A) and POLY_(B) is a polyethyleneglycol.